Protease activity of thrombin inhibits angiogenesis

ABSTRACT

The present invention features pharmaceutical compositions and methods to inhibit angiogenesis, with implications to cancer therapy. These methods are based on the discovery that activated thrombin has antiangiogenic activity and that this antiangiogenic activity is at least in part, mediated through the activation of a class of thrombin receptors termed, Protease Activated Receptor (PAR). Pharmaceutical compositions and methods are also directed to a class of proteases which mediate this activation, particularly the urokinase plasminogen activator (uPA) polypeptide.

FIELD OF THE INVENTION

The invention relates to the use of thrombin and its effectors toinhibit neovascularization.

BACKGROUND OF THE INVENTION

Angiogenesis is the growth of new blood vessels. Angiogenesis occursnaturally in mammals for healing wounds and for restoring blood flow totissues after injury or insult. In females, angiogenesis also occursduring the monthly reproductive cycle (to rebuild the uterus lining, tomature the egg during ovulation) and during pregnancy, to build theplacenta. The process of angiogenesis is, in part, governed byangiogenesis-stimulating factors and negatively regulated byangiogenesis inhibitors. When angiogenic factors are produced in excessof angiogenesis inhibitors, neovascularization is favored. Conversely,when inhibitors are present in excess of stimulators, angiogenesis isstopped.

Angiogenesis-associated diseases include, but are not limited to cancer,including HIV Kaposi's sarcoma, rheumatoid arthritis, psoriasis,pyogenic granuloma, diabetic retinopathy, macular degeneration, cornealgraft neovascularization, hypertrophic scarring, angiofibroma,Osler-Weber syndrome, neovascular glaucoma, and scleroderma.

Considerable data point to the importance of angiogenesis both inprimary tumor growth as well as for metastases. Tumor angiogenesis is acomplex process that is controlled by a balance between angiogenesisactivators and inlubitors. Proangiogenic molecules include vascularendothelial growth factor, interleukin 8 (IL-8), and basic fibroblastgrowth factor (bFGF), among others. Angiogenesis inhibitors can bedivided into several classes. One group consists of antibodies toproangiogenic factors or their receptors, as well as small moleculeinhibitors of signaling pathways triggered by these agents. A secondclass consists of endogenous proteins, such as thrombospondin andplatelet factor-4. A third group, which has attracted considerableattention recently, includes fragments of endogenous proteins where theparent protein is devoid of antiangiogenic activity or is evenproangiogenic. Examples include fragments of the extracellular matrix(endostatin, restin, tumstatin, and canstatin), as well as fragments orconformational states of molecules involved in coagulation andfibrinolysis (angiostatin: the first four kringle domains of plasminogenand the antiangiogenic conformation of anti-thrombin III). Theantiangiogenic and anti-tumor activities of these molecules have alsobeen demonstrated in vivo. Though some of these antiangiogenic proteinfragments are in clinical trials, they are difficult to produce and arenot orally available.

SUMMARY OF THE INVENTION

The present invention features methods to inhibit angiogenesis, whichmay be used, for example, in cancer therapies. These methods are basedon the discovery that activated thrombin has antiangiogenic activity andthat this antiangiogenic activity is at least in part, mediated throughthe activation of a class of thrombin receptors termed, ProteaseActivated Receptor (PAR).

Accordingly, in a first aspect, the invention features a method for thetreatment of angiogenesis-associated diseases. The method includes thesteps of administering a therapeutic amount of a pharmaceuticalcomposition comprising a Protease-Activated Receptor (PAR) agonistcapable of binding directly to the PAR receptor.

In a second aspect, the invention features another method for thetreatment of angiogenesis-associated diseases. This method includes thesteps of administering a therapeutic amount of a compound which resultsin activation of a Protease-Activated Receptor (PAR), the method,however, excludes administering either tissue plasminogen activator(tPA) polypeptide or a urokinase plasminogen activator (uPA), where theuPA is capable of binding to the human uPA receptor (uPA-R) if eitherthe tPA or uPA is administered in combination with captopril.

In a desirable embodiment of the first and second aspect, theangiogenesis associated diseases include but are not limited to cancer,rheumatoid arthritis, psoriasis, pyogenic granuloma, HIV Kaposi'ssarcoma, diabetic retinopathy, macular degeneration, corneal graftneovascularization, and hypertrophic scarring. Preferably the inventionis directed to treating cancer.

In other desirable embodiments to the first and second aspect, theProtease Activated Receptors are the thrombin binding PARs, PAR-1,PAR-3, and PAR-4.

In still other desirable embodiments of the first and second aspect, thePAR receptors can be activated directly with polypeptide ligands to thePARs (e.g., SFLLRNPNDKYEPF, SFLLRN, SALLRN, GYPGKF, and SLIGKV) or bymonoclonal antibodies. Desirably, the monoclonal antibody is modulating,more desirably, the monoclonal antibody prevents receptorinternalization.

In yet still another desirable embodiment of the first and secondaspect, treatment may be administered in combination with an ACEinhibitor, preferably from the group consisting but not limited tocaptopril, enalapril, lisinopril, benazepril, fosinopril, ramipril,quinapril, perindopril, trandolapril, and moexipril.

In a third aspect, the invention features a pharmaceutical compositioncomprising (i) substantially pure PAR-agonist, the agonist being capableof binding directly to the PARs; and (ii) a pharmaceutically acceptablecarrier.

In a fourth aspect, the invention features a pharmaceutical compositioncomprising (i) a therapeutic amount of a compound which results inactivation of PARs, this composition, however, does not comprise eithertPA polypeptide or uPA that is capable of binding to the human uPAreceptor in the absence of additional active ingredients other thancaptopril; and (ii) a pharmaceutically acceptable carrier.

In desirable embodiments to the third and fourth aspect, the ProteaseActivated Receptors are the thrombin binding PARs, PAR-1, PAR-3, andPAR-4.

In still other desirable embodiments of the third and fourth aspect,compositions can include polypeptide ligands to the PARs (e.g.,SFLLRNPNDKYEPF, SFLLRN, SALLRN, GYPGKF, and SLIGKV) or by monoclonalantibodies to the PARs. Desirably, the monoclonal antibody ismodulating, more desirably, the monoclonal antibody prevents receptorinternalization.

In a fifth aspect, the invention features a method for the treatment ofangiogenesis-associated diseases, this method involves administering atherapeutic amount of a pharmaceutical composition comprising thrombinor prothrombin to a patient diagnosed with an angiogenesis associateddisease.

In a desirable embodiment to the fifth aspect, treatment also includesan anti-coagulant.

Still in another desirable embodiment to the fifth aspect, treatmentalso includes administering an ACE inhibitor, from the group consistingbut not limited to captopril, enalapril, lisinopril, benazepril,fosinopril, ramipril, quinapril, perindopril, trandolapril, andmoexipril.

In a sixth aspect, the invention features a method for the treatment ofangiogenesis-associated diseases, this method involves administering apharmaceutical composition comprising a compound that modulates PARbiological activity. The treatment does not however, compriseadministering either tPA polypeptide or uPA that is capable of bindingto the human uPA receptor if the treatment also involves administeringcaptopril.

In a desirable embodiment of the fifth and sixth aspect, theangiogenesis associated diseases include but are not limited to cancer,rheumatoid arthritis, psoriasis, pyogenic granuloma, HIV Kaposi'ssarcoma, diabetic retinopathy, macular degeneration, corneal graftneovascularization, and hypertrophic scarring. Preferably the inventionis directed to treating cancer.

In a desirable embodiment to the sixth aspect, the Protease ActivatedReceptors are the thrombin binding PARS, PAR-1, PAR-3, and PAR-4.

In still other desirable embodiments to the sixth aspect, the PARs canbe activated directly with polypeptide ligands to the PARs (e.g.,SFLLRNPNDKYEPF, SFLLRN, SALLRN, GYPGKF, and SLIGKV) or by monoclonalantibodies. Desirably, the monoclonal antibody is modulating, moredesirably, the monoclonal antibody prevents receptor internalization.

In yet still another desirable embodiment of the sixth aspect, treatmentmay be administered in combination with an ACE inhibitor, from the groupconsisting but not limited to captopril, enalapril, lisinopril,benazepril, fosinopril, ramipril, quinapril, perindopril, trandolapril,and moexipril.

In a seventh aspect, the invention features a method for identifyingcandidate compounds that modulate PAR biological activity, the methodincludes the steps of: (a) contacting a Protease-Activated Receptor to acandidate compound; and (b) measuring the binding of the compound to thePARS, wherein said binding identifies the candidate compound as acompound that is useful for modulating PAR biological activity.

In a desirable embodiment to the seventh aspect, the Protease ActivatedReceptors used in the screen are the thrombin binding PARS, PAR-1,PAR-3, and PAR-4.

In an eighth aspect, the invention features a method for the treatmentof angiogenesis-associated diseases. This method involves administeringa pharmaceutical composition comprising substantially pure urokinase(uPA) polypeptide. The urokinase polypeptide is incapable of binding tothe urokinase receptor, uPA-R.

In a ninth aspect, the invention features a method for the treatment ofangiogenesis-associated diseases. This method involves introducing atransgene encoding a uPA polypeptide, the uPA polypeptide beingincapable of binding to the uPA receptor, to a cell. The transgene isoperably linked to expression control sequences, and positioned forexpression.

In a desirable embodiment of the eighth and ninth aspect, theangiogenesis associated diseases include but are not limited to cancer,rheumatoid arthritis, psoriasis, pyogenic granuloma, HIV Kaposi'ssarcoma, diabetic retinopathy, macular degeneration, corneal graftneovascularization, and hypertrophic scarring. Preferably the inventionis directed to treating cancer.

In a desirable embodiment of the eighth and ninth aspect, treatment maybe administered in combination with an ACE inhibitor, from the groupconsisting but not limited to captopril, enalapril, lisinopril,benazepril, fosinopril, ramipril, quinapril, perindopril, trandolapril,and moexipril.

In another desirable embodiment of the eighth and ninth aspect, theurokinase polypeptide is a mammalian urokinase that is incapable ofbinding to the human urokinase receptor. Desirably, the urokinasepolypeptide is mouse, rat, or human in origin. More desirably, theurokinase polypeptide is from human and the urokinase polypeptide issubstantially identical to the human uPA polypeptide sequence andfurther comprises amino acid residue substitutions in the Ω-loop.Desirably, wherein said human uPA further comprises amino acidsubstitutions within the Ω-loop. In another desirable embodiment, any 2,3, 4, 5, 6, or all 7 amino acids of the Ω-loop may be substituted withanother amino acid, typically a non-conservative amino acid. Mostdesirably, the amino acid residue substitutions are at amino acidresidues 27, 29, and 30 of the sequence²⁴tyr-²⁵phe-²⁶ser-²⁷asn-²⁸ile-²⁹his³⁰trp in human,²⁴tyr-²⁵phe-²⁶ser-²⁷arg-²⁸ile-²⁹arg-³⁰arg in mouse, and²⁴tyr-²⁵phe-²⁶ser-²⁷ser-²⁸ile-²⁹arg-³⁰arg in rat.

In a tenth aspect, the invention features a method for the treatment ofangiogenesis-associated diseases. This method involves introducing atransgene encoding a PAR polypeptide, and this transgene is operablylinked to expression control sequences, and said transgene beingpositioned for expression.

In a desirable embodiment of the tenth aspect, the angiogenesisassociated diseases include but are not limited to cancer, rheumatoidarthritis, psoriasis, pyogenic granuloma, HIV Kaposi's sarcoma, diabeticretinopathy, macular degeneration, corneal graft neovascularization, andhypertrophic scarring. Preferably the invention is directed to treatingcancer.

In other desirable embodiments to the tenth aspect, the ProteaseActivated Receptors to be expressed are the thrombin binding PARs,PAR-1, PAR-3, and PAR-4.

In still another desirable embodiment of the tenth aspect, treatment maybe administered in combination with an ACE inhibitor, from the groupconsisting but not limited to captopril, enalapril, lisinopril,benazepril, fosinopril, ramipril, quinapril, perindopril, trandolapril,and moexipril.

In yet still another desirable embodiment of the ninth and tenth aspect,the transgene is operably linked to a tissue-specific expression controlsequence.

In an embodiment to any of the foregoing aspects, methods furthercomprise administering an additional antiproliferative agentsimultaneously or within 14 days of each other in amounts sufficient toinhibit the growth of the neoplasm.

In an eleventh aspect, the invention features a method for identifyingantiangiogenic molecules in serum plasma. The method includes the stepsof: (i) contacting said serum plasma with a tissue protease and an ACEinhibitor; (ii) depleting said plasma of angiostatin; (iii)chromatographically separating plasma fractions; and (iv) determiningangiogenic potential of said fraction. The inhibition of angiogenesis inthe preceding assay identifies a fraction as antiangiogenic.

In a desirable embodiment to the eleventh aspect, mammalian serum plasmais used.

In another desirable embodiment to the eleventh aspect, the tissueprotease is selected from a group consisting of urokinase, tissueplasminogen activator, and streptokinase.

In still another desirable embodiment to the eleventh aspect, the ACEinhibitor is selected from a group, which includes but is not limited tocaptopril, enalapril, lisinopril, benazepril, fosinopril, ramipril,quinapril, perindopril, trandolapril, and moexipril.

In yet still another desirable embodiment to the eleventh aspect, thefraction having antiangiogenic activity is further purified to allow foridentification.

In a twelfth aspect, the invention features a pharmaceutical compositioncomprising (i) a therapeutic amount of a uPA, wherein the uPA isincapable of binding to the uPA-receptor; and (ii) a pharmaceuticallyacceptable carrier.

In a desirable embodiment to the twelfth aspect, the uPA polypeptide ismammalian, desirably, mouse, rat, or human uPA. More desirably, theurokinase polypeptide is from human and further comprises amino acidresidue substitutions in the Ω-loop.

In another desirable embodiment, any 2, 3, 4, 5, 6, or all 7 amino acidsof the Ω-loop may be substituted with another amino acid, typically anon-conservative amino acid. Most desirably, the amino acid residuesubstitutions of are on amino acid residues 27, 29, and 30 of thesequence ²⁴tyr-²⁵phe-²⁶ser-²⁷asn-²⁸ile-²⁹his-³⁰trp in human,²⁴tyr-²⁵phe-²⁶ser-²⁷arg-²⁸ile-²⁹arg-³⁰arg in mouse, and²⁴tyr-²⁵phe-²⁶ser-²⁷ ser-²⁸ile-²⁹arg-³⁰arg in rat.

In another embodiment to the twelfth aspect, the pharmaceuticalcomposition of the twelfth aspect, are used for the treatment of anangiogenesis-associated disease. Desirably, the angiogenesis-associateddisease is cancer, and more desirably, the cancer is breast cancer.

In a final aspect, the invention features a method for treatingangiogenesis-associated diseases. This method involves administering apharmaceutical composition comprising substantially pure urokinase (uPA)polypeptide and a second therapeutic agent. The urokinase polypeptide isincapable of binding to the urokinase receptor, UPA-R. Desirably, thesecond therapeutic agent is an antiproliferative agent. Administrationof uPA polypeptides of the invention and the antiproliferative agent maybe given simultaneously or within 14 days of each other in amountssufficient to inhibit the growth of the neoplasm.

In a related embodiment to any of the foregoing aspects, pharmaceuticalcompositions may further comprise a second therapeutic agent, desirably,the second therapeutic agent is an antiproliferative agent.

By an “antiproliferative agent” is meant a compound that, individually,inhibits the growth of a neoplasm. Antiproliferative agents of theinvention include microtubule inhibitors, topoisomerase inhibitors,platins, alkylating agents, and anti-metabolites. Particularantiproliferative agents include paclitaxel, gemcitabine, doxorubicin,vinblastine, etoposide, 5-fluorouracil, carboplatin, altretamine,aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin,busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin,colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine,dactinomycin, daunorubicin, docetaxel, estramustine phosphate,floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea,ifosfamide, imatinib, interferon, irinotecan, lomustine,mechlorethamine, melphalen, 6-mercaptopurine, methotrexate, mitomycin,mitotane, mitoxantrone, pentostatin, procarbazine, rituximab,streptozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine,topotecan, trastuzumab, vincristine, vindesine, vinorelbine, verapamil,and the uPA octamer-capped peptide, A6.

By “ACE inhibitor” is meant an angiotensin converting enzyme inhibitor.ACE inhibitors can be selected from a group comprising, but not limitedto captopril, enalapril, lisinopril, benazepril, fosinopril, ramipril,quinapril, perindopril, trandolapril, and moexipril.

By “agonist” is meant a drug or other chemical that can combine with areceptor on a cell to produce a physiologic reaction typical of anaturally occurring substance.

By “assaying” is meant analyzing the effect of a treatment, be itchemical or physical, administered to whole animals or cells derivedthere from. The material being analyzed may be an animal, a cell, alysate or extract derived from a cell, or a molecule derived from acell. The analysis may be, for example, for the purpose of detectingaltered gene expression, altered RNA stability, altered proteinstability, altered protein levels, or altered protein biologicalactivity. The means for analyzing may include, for example, antibodylabeling, immunoprecipitation, phosphorylation assays, and methods knownto those skilled in the art for detecting nucleic acids andpolypeptides.

By “cancer” or “neoplasm” is meant a cell or tissue multiplying orgrowing in an abnormal manner. Cancer growth is uncontrolled andprogressive, and occurs under conditions that would not elicit, or wouldcause cessation of, multiplication of normal cells.

By “candidate compound” is meant a chemical, be it naturally-occurringor artificially-derived, that is assayed for its ability to modulate analteration in reporter gene activity or protein levels, by employing oneof the assay methods described herein. Test compounds may include, forexample, peptides, polypeptides, synthesized organic molecules,naturally occurring organic molecules, nucleic acid molecules, andcomponents thereof.

By “expression control sequences” or a “promoter” is meant a nucleicacid sequence sufficient to direct transcription. Also included in theinvention are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell type-specific,tissue-specific or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the native gene.Desirable promoters of the invention direct transcription of a proteinin an endothelial cell; such promoters include, without limitation,promoters from the following genes: flt-1, Tie-1, Tie-2,endosialin/Tem-1, endoglin, and ICAM-2. Yet another desirable promoterof the invention directs transcription of a protein in an embryonalcell.

By “modulating” is meant conferring a change, either by decrease orincrease, in the level of a receptor mediated response relative to thatobserved in the absence of either thrombin or PAR agonist ligand or testcompound interaction with the PAR receptor or of the urokinasepolypeptide with the urokinase receptor. Preferably, the change inresponse is at least 5%, more preferably, the change in response is 20%and most preferably, the change in response level is a change of morethan 50% relative to the levels observed in the absence of thrombin, PARagonist ligand, or test compound.

By “operably linked” is meant that a nucleic acid molecule and one ormore regulatory sequences (e.g., a promoter) are connected in such a wayas to permit expression and/or secretion of the product (i.e., apolypeptide) of the nucleic acid molecule when the appropriate molecules(e.g., transcriptional activator proteins) are bound to the regulatorysequences.

By “pharmaceutically acceptable carrier” is meant a carrier that isphysiologically acceptable to the treated mammal while retaining thetherapeutic properties of the compound with which it is administered.One exemplary pharmaceutically acceptable carrier is physiologicalsaline. Other physiologically acceptable carriers and their formulationsare known to one skilled in the art and described, for example, in“Remington: The Science and Practice of Pharmacy” (20th ed., ed. A. R.Gennaro A R., 2000, Lippincott Williams & Wilkins).

By “positioned for expression” is meant that the DNA molecule ispositioned adjacent to a DNA sequence, which directs transcription andtranslation of the sequence (i.e., facilitates the production of, e.g.,PAR polypeptide).

By “Protease-Activated Receptor” or “PAR” is meant a G protein-coupledtransmembrane protein receptor capable of recognizing and bindingspecifically to thrombin, whereby thrombin activates this receptor bycleaving an amino-terminal exodomain to unmask a new amino terminusfollowing binding to the receptor. The new amino terminus is able tobind intramolecularly (a ‘tethered ligand’) to the body of theextracellular region of the receptor to effect transmembrane signaling.Four PARs are known in the art of which PAR-1, PAR-3, and PAR-4 arethrombin receptors. PAR-1 is the prototypic member of this family, whichbelongs to the 7-transmembrane receptor super-family. PAR-1 polypeptideand nucleotide sequences can be found in the NCBI database under GenBankAccession No. XM084176.

PAR biological activity is effected upon thrombin binding and processingof the receptor. Activation of the PAR by its tethered ligand allowssignaling through members of the G_(12/13), G_(q), and G_(i) G-proteinfamilies resulting in platelet and leukocyte recruitment and vascularpermeability in the endothelium. PAR-1 is the prototype of this receptorfamily and has been demonstrated to be a high affinity thrombinreceptor. Thrombin cleavage of human PAR-1 results in the exposure of anew amino terminus commencing with the peptide sequence SFLLRNPNDKYEPF.Synthetic peptides to the first six amino acid residues of the tetheredligand sequences of PAR receptors have been shown to function asagonists to the receptors, independent of receptor cleavage. PAR agonistligands can be selected from a group comprising but not limited to thepolypeptides, SFLLRNPNDKYEPF, SFLLRN, SALLRN, GYPGKF, and SLIGKV. By“protein” or “polypeptide” is meant any chain of amino acids, regardlessof length or post-translational modification (for example, glycosylationor phosphorylation).

By “substantially identical” is meant a polypeptide or nucleic acidexhibiting at least 75%, but preferably 85%, more preferably 90%, mostpreferably 95%, or even 99% identity to a reference amino acid ornucleic acid sequence. For polypeptides, the length of comparisonsequences will generally be at least 20 amino acids, preferably at least30 amino acids, more preferably at least 40 amino acids, and mostpreferably 50 amino acids. For nucleic acids, the length of comparisonsequences will generally be at least 60 nucleotides, preferably at least90 nucleotides, and more preferably at least 120 nucleotides.

Sequence identity is typically measured using sequence analysis softwarewith the default parameters specified therein (e.g., Sequence AnalysisSoftware Package of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Thissoftware program matches similar sequences by assigning degrees ofhomology to various substitutions, deletions, and other modifications.Conservative substitutions typically include substitutions within thefollowing groups: glycine, alanine, valine, isoleucine, leucine;aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine.

By “substantially pure polypeptide” is meant a polypeptide that has beenseparated from the components that naturally accompany it Typically, thepolypeptide is substantially pure when it is at least 60%, by weight,free from the proteins and naturally occurring organic molecules withwhich it is naturally associated. Preferably, the polypeptide is apolypeptide that is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight, pure. A substantially purepolypeptide may be obtained, for example, by extraction from a naturalsource (e.g., a fibroblast) by expression of a recombinant nucleic acidencoding the polypeptide, or by chemically synthesizing the protein.Purity can be measured by any appropriate method, e.g., by columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A protein is substantially free of naturally associated components whenit is separated from those contaminants, which accompany it in itsnatural state. Thus, a protein which is chemically synthesized orproduced in a cellular system different from the cell from which itnaturally originates will be substantially free from its naturallyassociated components. Accordingly, substantially pure polypeptides notonly include those derived from eukaryotic organisms but also thosesynthesized in E. coli or other prokaryotes.

By a “therapeutic amount” is meant an amount sufficient to result in theinhibition of angiogenesis. It will be appreciated that there will bemany ways known in the art to determine the therapeutic amount for agiven application. For example, the pharmacological methods for dosagedetermination may be used in the therapeutic context.

By “transgene” is meant any piece of nucleic acid that is inserted byartifice into a cell, or an ancestor thereof, and becomes part of thegenome of the animal, which develops from that cell. Such a transgenemay include a gene, which is partly or entirely heterologous (i.e.,foreign) to the transgenic animal, or may represent a gene homologous toan endogenous gene of the animal.

By “urokinase plasminogen activator” or “urokinase-type plasminogenactivator” or “urokinase” or “uPA” is meant a serine protease,substantially identical to the nucleotide and polypeptide sequences ofGenBank Accession No. NM_(—)002658 (human), NM_(—)008873 (mouse), orNM_(—)013085 (rat). Urokinase is produced as a single chain inactive(with respect to proteolytic activity) proprotein (pro-uPA). Cleavage ofthe pro-uPA, producing a two-chain mature uPA, precedes activation.Other biological activities of urokinase plasminogen activator includespecific cleavage of plasminogen (converting it into plasmin),activation of intracellular signaling upon binding to cell surfacereceptors, among them, the uPA receptor (uPA-R). The polyfunctionalproperties of this protein are associated with its three-domainstructure. The N-terminal domain shares homology to epidermal growthfactor, the central region having a kringle domain, and a C-terminalproteolytic domain containing the serine protease active center. uPA iscausally involved in cancer progression, particularly in invasion andmetastasis. Studies have shown breast cancer patients whose primarycancer contains high levels of uPA have a significantly worse outcomethan patients with low levels.

By “urokinase plasminogen activator receptor” or “uPA-R” or “CD87” ismeant a glycosylphosphatidylinositol (GPI)-anchored glycoprotein,substantially identical to the nucleotide and polypeptide sequence ofGenBank Accession No. XM086017. It should be appreciated that urokinaseplasminogen activator polypeptide displays specificity to uPA-R.Structure-function studies have shown the amino terminal domain of uPAbinds to the uPA-receptor with high affinity. Structural determinationof binding has been shown to depend on amino acid residues 24 to 30(human uPA), and termed the Ω-loop.

Interspecies cross over of ligand-receptor binding is not observed(i.e., a murine or porcine uPA does not bind to the human uPA-receptorand conversely, a human uPA does not bind to mouse uPA-receptor). In adesirable embodiment, polypeptides of the invention include alterationsto human, mouse, and rat uPA at amino acid residues, 24 to 30 (theΩ-loop), specifically to the sequence²⁴tyr-²⁵phe-²⁶ser-²⁷asn-²⁸ile-²⁹his-³⁰trp in human,²⁴tyr-²⁵phe-²⁶ser-²⁷arg-²⁸ile-²⁹arg-³⁰arg in mouse, and²⁴tyr-²⁵phe-²⁶ser-27 ser-²⁸ile-²⁹arg-³⁰arg in rat. We have demonstratedthat a triple mutant of murine uPA incorporating the human amino acidresidue substitutions at positions 27, 29, and 30 (i.e., R27N, R29H, andR30W) has been shown to ablate binding of murine uPA to the mouse UPA-Rreceptor.

Other features and advantages of the invention will be apparent from thefollowing description of the desirable embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-I are photographs of cells showing the inhibition ofendothelial cell tube formation by ex vivo treatment of plasma. HUVECcells, plus 10% untreated or treated fresh frozen plasma (FFP) in fullendothelial cell medium, were plated on each of 48-well platespreviously coated with matrigel and incubated overnight at 37° C.Heparin (1 U/ml) was added to all FFP samples prior to plating. A)Untreated cells in endothelial medium. B) Normal tube formation withuntreated FFP (10%). C) Significant inhibition by FFP treated with rt-PAand captopril (20%). D) Treated FFP at 10%. E) Treated FFP at 1% F)rt-PA (final concentration: 1 μg/ml) and captopril in PBS (finalconcentration: 0.1 μM). G) rt-PA alone, 1 μg/ml (no plasma, compare toA). H) Captopril alone, 0.1 μM (no FFP, compare to A). I) Heparin alone(1 U/ml). Bar: 250 μm.

FIG. 2 is a bar graph showing a quantitative analysis of tube formationassay (from FIG. 1). A) Untreated cells in endothelial medium. B)Untreated FFP (10%). C) FFP treated with rt-PA and captopril (20%). D)Treated FFP at 10%. E) Treated FFP at 1%. F) rt-PA and captopril in PBS(no FFP). G) rt-PA alone. H) Captopril alone. I) Heparin alone (1 U/ml,compare to A).

FIGS. 3A-E are photographs of cells showing inhibition of angiogenesis,in vivo, by systemic administration of rt-PA and captopril. The matrigelplug assay was performed as explained in materials and methods. Sectionsof each matrigel plug were stained by H&E and examined by lightmicroscopy. The total number of microvessels containing red blood cellsfrom 10 high power fields were counted and averaged. A) Mice treatedwith both rt-PA and captopril significantly inhibited in vivoneovascularization compared to the untreated control (+bFGF and PBS),the captopril alone group and less significantly compared with the rt-PAalone group. Each column represents the mean±SE of 3 plugs/group. B-E)Representative light microscopic appearance of matrigel plugs (H&Estaining and 400× magnification). B) Unstimulated (no bFGF) controlshowing reduced microvessels in the plug. C) Marked neovascularizationis observed in the untreated stimulated group. D) Significant reductionin neovascularization in the plugs of mice treated with rt-PA andcaptopril. E) In contrast, plugs of mice treated with captopril alonedid not show major reduction of neovascularization as compared to thecombination treatment group. Arrowheads indicate microvessels.

FIG. 4 shows the inhibition of endothelial cell proliferation caused byplasma of the patient treated with rt-PA and captopril. The assay wasperformed as in materials and methods. Note that at 0 hour (before rt-PAinfusion began) proliferation was increased compared to baseline (noplasma control). Plasma obtained at two hours into the treatment and onthe following hours caused a significant decrease in endothelial cellproliferation. The inhibitory effects persisted up to 48 hours after thestart of the infusion (values represent the average of triplicateexperiments). *p<0.007 (0 h vs. 12 h).

FIGS. 5A-F are photographs showing the inhibition of HUVEC tubeformation by the plasma of a patient treated with rt-PA and captopril.The patient received four cycles of this treatment with the dose ofrt-PA increased in each cycle. Plasma obtained at 4 hrs into theinfusion was used for this assay. The assay was performed as inmaterials and methods, except endothelial basal medium with 1% FCS wasused. A) Normal tube formation induced by the patient's plasma beforethe treatment. B) Mild inhibition of tube formation was observed afterthe first rt-PA dose. The effects were more marked when higher doses ofrt-PA were used. C) Second dose. D) Third dose. E) Fourth dose. F)Quantitative analysis of the above experiment (total tube length). Bar:250 μm.

FIGS. 6A-E are photographs of the strategies used to evaluate thecontribution of angiostatin on the antiangiogenic effects of treatedFFP. A) Treatment of HUVEC cells with angiostatin at 10 μg/ml. B)Affinity removal of angiostatin using lysine-Sepharose did not reducethe antiangiogenic effects of treated FFP (lysine flow through). C) Thelysine-bound fraction had a mild inhibitory effect on tube formation. D)Immunodepletion using a monoclonal antibody against human angiostatindid not affect the antiangiogenic activity of treated plasma. Bar: 250μm. E) Western blot using anti-angiostatin antibody (polyclonal againstkringle 1) showing increased levels of angiostatin by treated plasma andeffective removal by affinity chromatography and immunoprecipitation.Lane 1. Pure human angiostatin (kringles 14). Lane 2. Untreated FFP.Lane 3. Treated FFP. Lane 4. Lysine-Sepharose flow-through. Lane 5.Immunodepleted treated FFP.

FIGS. 7A-D are photographs of cells following fractionation of treatedFFP using a 3-step gradient on a Q-Sepharose column. Treated plasma wasapplied to the column. Three fractions were obtained (see text), andtheir activities were tested in the matrigel tube formation assay. A)Flow-through/300 mM NaCl wash had no antiangiogenic effects in vitro. B)400 mM NaCl fraction had significant antiangiogenic effects. C) 1000 mMNaCl wash showed no significant inhibition on angiogenesis. Bar: 250 μm.D) Western blot using anti-human angiostatin showed that theantiangiogenic fraction of the Q Sepharose column contained minimalamount of angiostatin-related proteins. Lane 1 Pure human angiostatin(kringles 14). Lane 2. Flow through and 300 mM NaCl wash contained mostof the angiostatin in the treated FFP. Lane 3. Fraction eluted at 400 mMNaCl contained minimal angiostatin. Lane 4. Fraction eluted at 1000 mMNaCl had no angiostatin.

FIGS. 8A-F are photographs of cells following immunodepletion (IP) ofplasma obtained from the patient treated with rt-PA and captopril.Plasma obtained at 4 and 8 hours into the rt-PA infusion wasimmunodepleted using a monoclonal antibody against angiostatin. A)Pre-treatment plasma stimulated tube formation. B) Four-hour plasmabefore depletion inhibited tube formation. C) The effect was retainedafter IP. The same effects were observed using the 8-hour plasma before(D) and after (E) IP. Bar: 250 μm. F). Western blot of patient's plasmabefore and after immunoprecipitation (IP) demonstrating effectiveremoval of angiostatin. Lane 1. Four-hour plasma pre IP. Lane 2.Four-hour plasma post-IP. Lane 3. Eight-hour plasma pre IP. Lane 4.Eight-hour plasma post-IP. Lane 5. Plasma before rt-PA infusion.

FIG. 9 shows a 4-15% gradient SDS-PAGE gel showing the progress ofprotein purification. Approximately 10-20 μg of proteins were loadedonto each lane. Proteins were visualized by Coomassie Brilliant Bluestaining.

FIGS. 10A-J are photographs of cells treated with fractions derived fromthe purification of antiangiogenic activity in tPA/captopril-treatedhuman plasma. (A) Negative control; (B) In vitro tPA/captopril-treatedplasma; (C) HiTrap QXL flow-through fraction; (D) HiTrap QXL 400-mM NaClfraction; (E) HiTrap QXL 1000-mM NaCl fraction; (F) HiTrap Blueflow-through fraction; (G) HiTrap Blue 1.5-M NaCl fraction; (H) HiTrapBlue 2-M guanidine hydrochloride fraction; (1) Ni-NTA flow-throughfraction; (J) Ni-NTA 200-mM imidazole fraction.

FIGS. 11A-L are photographs of cells showing the effects of the proteaseactivity of thrombin on endothelial cell tube formation in vitro. (A)Negative control; (B) 5 μg/ml prothrombin; (C) 10 μg/ml prothrombin; (D)1 U/ml thrombin; (E) 5 U/ml thrombin; (F) 10 U/ml thrombin; (G) 10 U/mlthrombin with 200 U/ml lepirudin; (H) 10 g/ml prothrombin with 200 U/mllepirudin; (I) 10% (v/v) tPA/captopril-treated plasma; (J) 10% (v/v)tPA/captopril-treated plasma with 200 U/ml lepirudin; (K) 10% (v/v)untreated plasma with 200 U/ml lepirudin; (L) 200 U/ml lepirudin.

FIGS. 12A-D are photographs of cells showing the effects ofthrombin-receptor-activating peptide (TRAP) on endothelial cell tubeformation in vitro. (A) Negative control; (B) 1 μM; (C) 10 μM; (D) 100μM of TRAP respectively.

FIGS. 13A-E are photographs showing Matrigel-plug assays to evaluateangiogenesis in vivo. (A-D): H&E stained sections of matrigel plugsexcised from mice 10 days post implantation. (A) No bFGF; (B) 250 ng/mlbFGF; (C) 250 ng/ml bFGF and 10 ρg/ml prothrombin; (D) 250 ng/ml bFGFand 250 μM TRAP. Arrowheads point to red-blood-cell containingmicro-vessels. (E) The results of the matrigel-plug assays depicted in ahistogram.

FIGS. 14A-F are photographs of cells showing the effects of activationof various PARs on endothelial cell tube formation in vitro. (A)Negative control; (B) 50 μM SFLLRN; (C) 100 μM SFLLRN; (D) 100 μMSALLRN; (E) 100 μM SLIGKV; (F) 100 μM GYPGKF.

FIG. 15 is a bar graph showing the effects of tissue proteaseoverexpression on tumor cell growth. Metastatic breast cancer cell lines(4T1) stably expressing tPA (tpa), wildtype uPa (upa), or acatalytically active, receptor binding mutant of uPA (uPA R27N, R29H,and R30W; upa mut) were implanted into BALB/c mice. Tumor growth wasmeasured every other day for 34 days. The data collected is displayed asa line graph. Results for tumor growth were measured as a function oftime. N=12 in each group of mice; EV=empty vector.

DETAILED DESCRIPTION

We have observed that plasma serum treated with an ACE inhibitor and aprotease, unlocks an angiogenic potential that can be exploited for thetreatment of angiogenic disorders. We have discovered one of the factorsnecessary for clotting, Factor-II (widely known as thrombin), possessesthis antiangiogenic potential.

The antiangiogenic potential of thrombin is at least in part mediatedthrough the molecular interactions of thrombin to its receptor, PAR-1, aG-protein coupled receptor. Binding of thrombin to its receptor leads tothe cleavage of an amino terminus of the receptor and consequentlyexposing a polypeptide sequence capable of intramolecularly associatingwith itself. Soluble versions of this ‘tethered ligand,’ upon binding toits receptor, also effect antiangiogenic activity in human endothelialcells.

Thus by introducing thrombin or ligands and mimetics that activate thethrombin receptor in regions of the body affected by angiogenicassociated diseases, we can modulate the mechanisms involved in localneovascularization.

EXAMPLE 1 In Vitro and In Vivo Induction of Antiangiogenic Activity byPlasminogen Activators and Captopril

In vitro exposure of human fresh frozen plasma to rt-PA and captoprilinduced significant in vitro antiangiogenic activity as assessed by thematrigel tube formation assay (FIG. 1 and FIG. 2). Pharmacokineticstudies have shown that plasma concentrations of 0.1 to 1 μM areachieved by doses of captopril of 25 to 37.5 mg three times a day.Plasma concentrations of tissue plasminogen activator in healthyvolunteers and patients treated for myocardial infarction are in therange of 0.5 to 1.8 μg/ml receiving doses of 0.004 mg/kg/min. Theconcentrations used in our assays are within this range. The in vitrofindings were extended to the in vivo setting by treatment of a patientwith metastatic sarcoma with captopril and low dose rt-PA. A potentantiangiogenic effect was induced in her plasma (FIGS. 4 and 5). Thedemonstration that the treated patient's plasma inhibited endothelialcell proliferation and capillary tube formation suggests thatbiologically relevant antiangiogenic effects can be induced atclinically tolerable doses of rt-PA and captopril. Moreover, the findingthat systemic administration of rt-PA and captopril into mice decreasedneovascularization in the matrigel plug assay is further indication thatthe effects induced by the treatment may have important biological invivo relevance.

A novel finding was that the observed antiangiogenic effect from FFPexposed to rt-PA and captopril was not primarily due to the generationof angiostatin. Several lines of evidence speak to this. First, pureangiostatin at concentrations of 10 μg/ml and 50 μg/ml did notsignificantly inhibit tube formation (FIG. 7A and FIG. 7B). Second,affinity removal of angiostatin from FFP exposed to rt-PA and captoprildid not remove the antiangiogenic activities (FIG. 7C). Third, treatedFFP retained the tube formation inhibitory activities after angiostatinimmunodepletion (FIG. 7D). Finally, fractionation of treated plasmademonstrated that the antiangiogenic fraction contained little or noangiostatin and the fraction that contained angiostatin had nosignificant inhibitory activity on in vitro angiogenesis (FIG. 8).

That angiostatin did not play a major role in the antiangiogenic effectsof the treated plasma was unexpected. The data provided in this studysuggest that other antiangiogenic molecules are generated as a result ofthe rt-PA/captopril treatment of plasma. These appeared to be separablefrom and more potent than angiostatin. That angiostatin did not have asignificant inhibitory effect on tube formation may be related to theconditions of the assays employed. In our assays (for ex vivo treatmentof plasma), we resuspended the cells in full endothelial growth media,with growth factors such as VEGF, bFGF, hEGF, IGF-1, etc. Reports thatdemonstrated inhibition of tube formation by angiostatin employed a lessrich medium of either VEGF alone, bFGF alone, or low serumconcentrations.

Another interesting finding is the duration of the antiangiogeniceffects in the plasma of the patient after treatment. rt-PA is rapidlycleared from plasma, however, the endothelial antiproliferative effectsof the patient's plasma persisted up to 36 hours after the infusion wasstopped (FIG. 4). This evidence suggests that the effects noted were notmediated directly by rt-PA but newly generated molecule(s) with arelatively long half-life.

Role of ACE inhibitors. The results from our in vivo assays suggest thatthere may be a small but significant contribution of captopril to theantiangiogenic effects of rt-PA treatment (compare the matrigel plugmicrovessel counts from mice treated with rt-PA and captopril vs. thecounts of mice treated with rt-PA alone). There are several plausibleexplanations. In addition to stimulating generation of angiostatin,captopril may have antiangiogenic effects by itself that could beadditive or synergistic to the effects of rt-PA. Several studies havereported the antiangiogenic properties of captopril. However, theconcentrations of captopril used for in vitro inhibition of angiogenesiswere in the millimolar range. The antiangiogenic effects of captoprilcould be due to its ability to regulate extracellular (EC) tPA and PAI-1production. There is evidence that the angiotensin-converting enzyme(ACE) plays an important role in regulating the fibrinolytic cascade byvirtue of its endothelial localization and its roles in activatingangiotensin and degrading bradykinin. Bradykinin is one of the mostpotent stimuli regulating the synthesis and secretion of tPA, andangiotensin appears to be an important regulator of PAI-1 production.Inhibiting EC ACE would theoretically down-regulate expression of PAI-1and up-regulate expression of tpA. Captopril down-regulates expressionof PAI-1 in vitro and in vivo in patients with acute myocardialinfarction.

Finally, there maybe an additional component of the antiangiogenicaction of rt-PA that would escape detection by our in vitro endothelialassays utilizing plasma. The generation of plasmin (from plasminogen) inthe tumor microenvironment by rt-PA could trigger a series ofproteolytic events leading to degradation of the tumor matrix. Duringthe initial stages of tumor angiogenesis, fibrin formation anddeposition are important to set a favorable environment for new vesselformation. rt-PA may activate fibrin bound plasminogen and enhancedegradation of tumor stroma (and fibrin in particular), potentiallyimpeding neovascularization. In view of tissue plasminogen activator'sability to promote plasmin formation when bound to plasminogen andfibrin, rt-PA administration could represent a “targeted” strategy topreferentially inhibit angiogenesis in the tumor microenvironment. Thishypothesis may explain why tumors that overexpress tissue plasminogenactivator are associated with less metastases in preclinical models andwith a better prognosis (improved metastasis free survival and overallsurvival) in patients with breast cancer and melanoma.

Results

Ex Vivo Treatment of Human Plasma Inhibits in Vitro Angiogenesis.Untreated or treated fresh frozen plasma (FFP) was added to 4×10⁴ HUVECcells to 10% (v/v) and then seeded in each of a 48-well matrigel coatedplate. Untreated cells in full growth medium were used as a negativecontrol. After 12-16 hours of incubation, treated FFP, but notuntreated, exhibited a striking inhibition of EC tube formation (seeimages in FIG. 1, and quantitative analysis in FIG. 2). rt-PA (FIG. 1G)or captopril (FIG. 1H), alone or in combination (FIG. 1F), in theabsence of plasma, did not cause significant inhibition of tubeformation. The inhibitory effects decreased as the concentration oftreated plasma was reduced (FIGS. 1C, 1D and 1E). Similar data wasobtained with plasma from three patients with cancer exposed in vitro tort-PA and captopril.

In Vivo Antiangiogenic Effects of systemic administration of rt-PA andcaptopril. Mice were injected with matrigel and treated as described inmaterials and methods. The matrigel plugs of all groups of mice (exceptthe unstimulated control) contained bFGF as a proangiogenic stimulant.None of the treatment groups developed any significant adverse eventfrom the treatments. At day 10, mice were sacrificed, and the plugs wereanalyzed Only microvessels that contained red blood cells were counted.Plugs in mice treated with the both rt-PA and captopril hadsignificantly less microvessel counts than the groups treated with PBS,rt-PA alone, or captopril alone (FIG. 3A). Representative sections (H&Estains) of the matrigel plugs are shown in FIGS. 3B to 3E.

Induction of Antiangiogenic Effects in the Plasma of a Patient Treatedwith rt-PA and Captopril The patient's plasma obtained during treatmentwas used to perform EC proliferation and matrigel tube formation assays.At the second rt-PA dose level (0.02 mg/kg/hr), we observed asignificant inhibition of HUVE cell proliferation with plasma obtainedduring treatment, compared to her pretreatment plasma (FIG. 4). Thiseffect lasted for up to 48 hours after the start of the infusion anddecreased significantly by 144 hours. Moreover, her plasma (during theinfusion) caused a significant inhibition of HUVEC tube formation afterthe second dose level (FIG. 5). The antiangiogenic effect was mildlyenhanced with increasing doses of rt-PA infusion (FIG. 5).

Contribution of Angiostatin to the Antiangiogenic Effects of TreatedPlasma. Previous publications have demonstrated in vitro conversion ofplasminogen to angiostatin after incubation of plasminogen withplasminogen activators and sulfhydryl donors. In order to determine thecontribution of angiostatin to the antiangiogenic effects of treatedFFP, a comparison was made between human angiostatin and treated FFP.Angiostatin at 10 μg/ml (FIG. 6A) or 50 μg/ml did not significantlyinhibit tube formation in the matrigel assay. In comparison, asignificant inhibitory effect was achieved with 10% treated FFP (FIG.1B), which theoretically should contain approximately 10 μg/ml ofangiostatin, assuming full conversion of plasminogen (200 μg/ml in 100%plasma) to angiostatin.

Next, affinity chromatography using lysine-Sepharose was performed ontreated FFP. A western blot analysis demonstrated removal of angiostatinfrom the treated plasma (FIG. 6E, compare lane 4 vs. lane 3). The tubeformation inhibitory effects of plasma depleted of angiostatin (flowthrough) were retained (FIG. 6C) and appeared similar to treated FFPbefore affinity removal of angiostatin. The lysine bound fraction had avery mild inhibitory effect on tube formation (FIG. 6B).

To further confirm this observation, treated plasma was immunodepletedof angiostatin using monoclonal antibodies against human angiostatin.Successful removal of angiostatin was demonstrated by western blotanalysis (FIG. 6E, lane 5). The antiangiogenic effects of treatedplasma, as assessed by the matrigel tube formation assay, were alsoretained after angiostatin immunodepletion (FIG. 6D).

Finally, we performed ion exchange chromatography using a Q-Sepharosecolumn on the treated plasma. Three fractions were obtained (flowthrough after loading at 150 mM combined with a wash at 300 mM NaCl,eluate at 400 mM NaCl, and wash at 1 M NaCl). Each fraction was used totreat HUVEC cells for the matrigel tube formation assay. Theantiangiogenic effect was present in the fraction eluted at 400 mM NaCl(FIG. 7B). The flow through (FIG. 7A) and the 1 M NaCl (FIG. 7C)fractions did not have any significant effect on in vitro angiogenesis.Western blot analysis of the above fractions showed that angiostatin waspresent on the flow through and very minimally in the 400 mM fraction(FIG. 7D). Thus, there was a clear dissociation between the presence ofangiostatin and antiangiogenic activity. Antiangiogenic Effects of thePlasma from the Treated Patient were not Completely due to Angiostatin.The next logical question was whether the antiangiogenic effects seen invivo, from the plasma of the patient treated with rt-PA and captopril,were predominantly due to angiostatin. We performed angiostatinimmunodepletion as described above. Plasma obtained from two time pointsduring the treatment was used for immunodepletion (obtained at week 2 oftreatment and at 4 and 8 hours into rt-PA infusion at 0.02 mg/kg/hr). Awestern blot shows angiostatin to be increased compared to pre-treatmentand that it was successfully removed after immunodepletion (FIG. 8F).The inhibitory effects on tube formation is preserved on theangiostatin-depleted plasma (compare FIGS. 8B with 8C and 8D with 8E).

Experimental Procedures

Reagents. rt-PA (Genentech, San Francisco, Calif.) and captopril(Sigma-Aldrich Research, St. Louis, Mo.) were diluted in sterilephosphate buffer saline and used for the bioassays. Heparin (Elkins-SinnInc, Richmond, Va.) or lepirudin (Aventis Pharmaceuticals, Kansas City,Mo.) was added to FFP or patient's plasma to prevent clot formation.Matrigel (Collaborative Biomedical Products, Bedford, Mass.), a basementmembrane preparation from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma,was used at 7 mg/ml for in vitro angiogenesis (tube formation) assaysand at 10 mg/ml for in vivo (matrigel plug) assays (see below). Basicfibroblast growth factor was purchased from Peprotech; Rocky Hill, N.J.The cell proliferation reagent WST-1 (Roche; Indianapolis, Ind.) wasused for proliferation assays. WST-1, a tetrazolium salt, is cleaved toformazan by mitochondrial dehydrogenases in viable cells. A murinemonoclonal antibody against human angiostatin (Calbiochem; San Diego,Calif.) was used for western blotting and immunodepletion. A rabbitpolyclonal antibody against mouse angiostatin (Affinity Bioreagents Inc;Golden, Colo.) that cross-reacts with human angiostatin was used forwestern blotting. Human angiostatin (kringles 14) was obtained fromCalbiochem (San Diego, Calif.). Case Report. EB is a 46 year-old femalewith a history of metastatic malignant fibrous histiocytoma. She has hadmultiple recurrences following surgical resections (pulmonary, hepatic,and subcutaneous nodules), radiation therapy, and thalidomide treatment.She refused standard chemotherapy. The patient was screened for bleedingdisorders and for brain metastases and signed an informed consent. Shestarted taking captopril at 25 mg p.o. three times a day. One weeklater, she received a 12-hour intravenous infusion of rt-PA. The patientreceived a total of four 12-hour intravenous infusions of rt-PA during afour-week period, with increasing doses each week, starting at 0.015mg/kg/hr, 0.02, 0.03, and 0.035 mg/kg/hr (fourth dose). Blood was takenfor coagulation tests (thrombin time, fibrinogen, PT, and aPTI) and forbioassays at varying time points before, during, and following theinfusion. Lepirudin (5 μg/ml) was added to the patient's plasma used forbioassays to prevent clot formation. Neither during nor after theinfusions did the patient experience any significant adverse reactions.

Human Plasma. Outdated fresh frozen plasma (FFP) was obtained from BethIsrael Deaconess Medical Center's blood bank. Blood from theabove-described patient with cancer was collected from a peripheral veininto citrated tubes. The blood was immediately centrifuged at 3750 rpmfor 10 minutes. Both fresh frozen plasma and the patient's plasma werefilter-sterilized (0.2 μm sterile filters, Millipore Corporation;Bedford, Mass.) and then stored at −20° C. for future use. Captopril (1μM) and rt-PA (10 μg/ml) were added to 1 ml of FFP and incubated for 3hours at 37° C. before performing the bioassays.

Cell Culture. Human umbilical vein endothelial (HUVEC) cells wereobtained from Clonetics (San Diego, Calif.) and used between passages 3and 5. They were maintained in EGM2-MV medium (BioWhittaker;Walkersville, Md.) that contains endothelial basal medium (EBM-2),supplemented with 5% fetal bovine serum, gentamicin, amphotericin B,hydrocortisone, ascorbic acid, and the following growth factors: VEGF,bFGF, hEGF, and IGF-1. Cells were grown at 37° C., in a 100% humidifiedincubator with 5% CO₂. Cells were grown to 80-90% confluency, harvestedwith trypsin, and resuspended to the cell density required for eachassay.

In Vitro Angiogenesis (matrigel tube formation) Assay. Unpolymerizedmatrigel (7 mg/ml) was placed in the wells (100 μl/well) of apre-chilled 48-well cell culture plate and then incubated at 37° C. for30-45 minutes. HUVEC cells were harvested in trypsin and resuspended inEC medium (4×10⁴ in 300 μl). Cells were treated with the differentagents before plated onto the matrigel-coated plates. After 12 hours ofincubation, tube formation was observed through an invertedphotomicroscope (Nikon; Tokyo, Japan). Microphotographs of the center ofeach well at low power (40×) were taken with a SPOT camera (DiagnosticInstruments Inc; Sterling Heights, Mich.) and the aid of an imagingcapture software (Compix Inc Imaging Systems; Township, Pa.). Themicrophotographs were quantitatively analyzed (total tube length) withthe Simple PCI imaging analysis software (Compix). Untreated HUVEC cellsin EC medium were used as a negative control, and actinomycin D(Sigma-Aldrich Research) (7.5 μg/ml) was used as a positive (inhibitory)control.

Cell Proliferation Assay. Cells (4×10³/well, in a total volume of 100μl) were seeded into each well of a 96-well plate and maintained in theappropriate basal medium with 1% fetal bovine serum, penicillin, andstreptomycin. Cells were suspended in 1% FBS and treated with the activeagents and incubated at 37° C. for 72 hours. We observed that plasma wasa potent stimulant of EC proliferation, and therefore we did not use anyadditional stimulant of proliferation (VEGF and/or bFGF) on theseassays. At the end of the specified period, WST-1 (10 μl) was added toeach well and incubated at 37° C. for three hours. After incubation,absorbance at 450 nm was determined using a microplate reader (Bio-Rad;Hercules, Calif.). The experiments were performed in triplicate, and thefigures presented represent the average of triplicate experiments.Statistical analysis was performed using a t-test analysis (paired twosample for means analysis).

In Vivo Angiogenesis (Matrigel Plug) Assay. The matrigel-plug assay wasperformed as described in Maeshima et al., (Maeshima et al., J Biol Chem275:21340-8, 2000; Maeshima et al., J Biol Chem 276:31959-68, 2001) withmodifications as follows. All animal studies were reviewed and approvedby the animal care and use committee of Beth Israel Deaconess MedicalCenter and are in accordance with the guidelines of the department ofHealth and Human Services. Five to six week-old male C57/BL6 mice (TheJackson Laboratories; Bar Harbor, Me.) were injected subcutaneously atthe left lower abdominal wall with 0.5 ml of unpolymerized matrigelsupplemented with 500 ng/ml of basic fibroblast growth factor for thestimulated controls and treatment groups and with equivalent volume ofsterile PBS for the unstimulated (no b-FGF) control group. Mice (3 pergroup) were treated for 10 days with: 1) rt-PA (60 μgsubcutaneously/day) and captopril (150 μg intraperitoneally/day). 2)Subcutaneous rt-PA alone with equivalent volume of intraperitoneal (IP)PBS. 3) IP captopril (150 μg) with equivalent volume of subcutaneousPBS. 4) Equivalent volumes of PBS (subcutaneously andintraperitoneally). At day 10, mice were sacrificed; the matrigel plugswere removed and fixed in 4% paraformaldehyde, embedded in paraffin,sectioned, and H&E stained. Sections were examined by light microscopy,and the total number of blood vessels from 10 high power fields (400×magnification) were counted in a blinded fashion. Results shownrepresent the average of counts from three matrigel plugs per group.

Affinity Chromatography. Lysine-Sepharose (Pharmacia; Piscataway, N.J.)chromatography was used to separate angiostatin from the rest of thetreated FFP. Briefly, a lysine-Sepharose column (6 ml) was made as permanufacturer's recommendations. The procedure was performed at 4° C.Treated plasma (10 ml) was loaded onto the column pre-equilibrated with50 mM sodium phosphate (pH 7.5), followed by successive washes with 50mM sodium phosphate (pH 7.5) (10 volumes), PBS (5 volumes), and 0.5 MNaCl (5 volumes). Retained proteins were eluted using epsilonaminocaproic acid (Sigma-Aldrich Research, St. Louis, Mo.) at 200 mM inwater. The eluted protein was dialyzed (dialysis membrane with amolecular weight cutoff of 3000 obtained from Pierce Chemical Company;Rockford, Ill.) against PBS (4 L) for 48 hours, concentrated to theoriginal volume of the plasma, filter sterilized, and stored at −20° C.for future use.

Angiostatin Immunoprecipitation of Plasma. Treated plasma (200 μl) wasincubated overnight with a monoclonal antibody against human angiostatin(32 μg/ml) and rocked at 4° C. The next day, protein A+G agarose (50 μl)was added and rocked for 2 hours at 4° C. The samples were centrifuged(12,000 rpm for 5 minutes), and the supernatant (IP'd plasma) was storedat −20° C. for future use.

Fractionation of Treated Plasma. A series of small-scale anion exchangechromatographic steps was initially employed to optimize separation ofangiostatin from the antiangiogenic activities generated in FFP by thert-PA and captopril treatment. Treated FFP (1 ml) was exchanged intobuffer A (10 mM Tris HCl pH 7.4)/50 mM NaCl by a NAP-10 column(Pharmacia). The sample was applied onto a 1-ml HiTrap QXL (Pharmacia)pre-equilibrated with buffer A/50 mM NaCl at 1 ml/min. The column waswashed with the start buffer until the absorbance at 280 nm returned tobaseline. Proteins were eluted by a step gradient of NaCl (50-mMincrements) until 500 mM NaCl was reached. The column was then washedwith buffer A/1M NaCl. All fractions were concentrated and exchangedinto 1×PBS before testing for activities. The antiangiogenic activitieswere eluted between 300 and 400 mM NaCl fractions. Preparative-scaleseparation was performed by applying treated FFP onto a 20-ml HiPrep16/10 Q XL column (Pharmacia). The column was washed extensively withBuffer A/300 mM NaCl. Absorbed proteins were eluted from the columnsequentially with buffer A/400 mM NaCl and buffer A/1 M NaCl. Allfractions were concentrated and exchanged into 1×PBS and stored at −20°C. for further use.

SDS PAGE and Western Blot. Protein samples diluted with SDS/DTT wereseparated by 4-20% polyacrylamide gel electrophoresis (pre-cast gels,Bio-Rad). This was followed by electroblotting onto apolyvinylidenedifluoride (PVDF) membrane. After blocking with 2% BSA inTris-buffered saline/tween-20 (TTBS) for 1 hour, the PVDF membrane wasincubated overnight with the polyclonal angiostatin antibody (2 μg/m).After washing with TTBS, the membrane was incubated with a horseradishperoxidase-conjugated secondary antibody (Amersham Corporation;Arlington Heights, Ill.; 1:5000 dilution) for 1 hour. The protein bandswere detected using SuperSignal® West Pico Chemiluminescent Substrate(Pierce Chemical Company; Rockford, Ill.).

EXAMPLE 2 Protease Activity of Thrombin Inhibits Angiogenesis

We have discovered that treatment of whole human plasma with tPA incombination with captopril in vitro resulted in the generation of anantiangiogenic activity. However, the induced antiangiogenic moiety inthis treated plasma was unlikely to be angiostatin, as removal ofangiostatin by either immunodepletion or lysine Sepharose chromatographydid not diminish the antiangiogenic activity present in thetPA/captopril-treated plasma. Here we describe a series of steps topurify and identify the protein with this novel antiangiogenic activity.

We have developed an efficient three-column scheme. First, the treatedplasma is subjected to anion exchange chromatography, yielding afraction that contains most of the antiangiogenic activity (FIG. 10D)but accounts for <2% of the input proteins. Interestingly, the potencyof this fraction (and the active fractions from the later columns) ismore than that of the tPA/captopril-treated plasma prior to purification(compare FIGS. 10B and 10D). We believe this difference may reflect thefact that whole plasma contains angiogenic factors that are not presentin the active fractions.

Antiangiogenic activity is further purified by Cibacron BlueF3G-A-affinity (Blue Sepharose) and Ni-NTA chromatography. The BlueSepharose column effectively removes about 60% of the bulk proteins(FIG. 9, lane 6). Most importantly, under our conditions, the Ni-NTAcolumn essentially absorbs all of the remaining contaminating proteins(FIG. 9, lane 8) while the active species flows through into the‘flow-through’ fraction (FIG. 101).

The purified antiangiogenic protein was submitted to protein sequencingby MALDI-TOF. The spectral data revealed that the antiangiogenic proteinpurified from in vitro tPA/captopril-treated plasma to be prothrombinwith a molecular weight of 71904.7. Since it was likely that addition oftPA/captopril in plasma under these conditions initiated a cascade ofproteolysis, we wanted to determine whether prothrombin wasproteolytically altered during the treatment and thus becameantiangiogenic. To address this possibility, a sample of commerciallypurchased prothrombin was submitted for analysis by mass spectroscopyand was determined to have a molecular weight of 71441, which indicatedthat it was actually smaller than the prothrombin purified from thetPA/captopril-treated plasma. This subtle difference in the molecularweights may reflect differences in glycosylation or extent of gammacarboxylation at the Gla domain of prothrombin. It is also possible thatsome protein degradation occurred in the commercially available protein.However, the minor difference in the molecular weights apparently wasnot related to prothrombin's antiangiogenic activity, because thecommercial prothrombin was also antiangiogenic (FIGS. 11B and 11C) andappeared to have the same potency as the prothrombin purified from thetPA/captopril-treated plasma. Furthermore, we also showed thatprothrombin purified from untreated human plasma using the samepurification scheme was also antiangiogenic. Since prothrombinactivation normally leads to clot formation, the antiangiogenic activityof thrombin in whole plasma was not previously evaluated. We showed thataddition of tPA and captopril unmasked this activity in whole plasma. Wehypothesize that this induction of activity may be due to the decreasein the clotting potential of the plasma post treatment. Consistent withthis notion, when fibrinogen was removed by either anionic orhydrophobic interaction chromatography, the fraction containingprothrombin was potently antiangiogenic. Furthermore, fractions ofuntreated plasma that were devoid of prothrombin were not antiangiogenicand could not be rendered antiangiogenic by the tPA/captopril treatment.

Collectively, our data suggest that prothrombin is antiangiogenic invitro and in vivo. Although normally suppressed in plasma, this activityof prothrombin was unmasked by treatment with tPA/captopril in vitro.

Structurally, prothrombin can be divided into four domains: a Gladomain, a kringle 1 domain, a kringle 2 domain, and a serine proteasedomain. The Gla and kringle 1 domains together are often referred as thefragment 1 of prothrombin, whereas the kringle 2 domain is also calledfragment 2. Previously it was reported that both fragments 1 and 2 ofprothrombin inhibited endothelial cell proliferation in vitro andangiogenesis in the chorioallantoic membrane of chick embryo. In thisreport, we examined the contribution of the protease activity ofthrombin to the induced antiangiogenic activity in tPA/captopril-treatedplasma. We showed that thrombin significantly inhibits endothelial celltube formation at 10 U/ml (FIG. 11F). This effect was completely blockedby the addition of lepirudin (FIG. 11G), a specific thrombin inhibitor.Significantly, the inhibitory effect of prothrombin (FIG. 11C) was alsoblocked by lepirudin (FIG. 11H). Since prothrombin is devoid ofproteolytic activity, our data suggest that prothrombin becameproteolytically active when incubated with endothelial cells on matrigelin vitro. Western blot analyses showed that prothrombin wasproteolytically cleaved into smaller fragments during the assay.

Based on our results, thrombin modulates angiogenesis. It is known thatthrombin directly affects endothelial cell functions that are regulatedduring the angiogenic process. For example, thrombin up-regulatesendothelial cell secretion of matrix metalloproteinase (MMP)-1 and -3,affects secretion of platelet-derived growth factor (PDGF), tumor growthfactor (TGF)-β1, and bFGF by endothelial cells, diminishes adhesion ofendothelial cells to extracellular matrix, promotes an increase inbasolateral deposition, and a decrease in apical release of theextracellular matrix proteins fibronectin, laminin, and collagens I andIV, induces endothelial cell contraction and vascular permeability,up-regulates expression of VEGF receptors (KDR and flt-1), andstimulates endothelial cell migration. In addition, it has beenpostulated that thrombin affects angiogenesis by activating gelatinaseA. Thrombin may also affect angiogenesis indirectly through plateletactivation. For example, platelets release cytokines including PDGF andVEGF and the angiogenesis inhibitor endostatin upon activation bythrombin.

Based on out results, we propose that thrombin has a bimodal effect onendothelial cell functions. We have observed a switch from stimulatoryto inhibitory, as the concentration of thrombin was increased onendothelial cells in culture. This biphasic property was also reportedwhen the effects of thrombin were assessed in either in vitroendothelial cell tube formation or in an in vivo chick chorioallantoicmembrane assay.

Results

Protein Purification. We sought to identity the active antiangiogenicingredient of tPA/captopril-treated plasma by column chromatography andused endothelial cell tube formation as a read-out. The primary reasonfor choosing this in vitro assay is that the effects of proteinfractions can be determined rapidly, usually in less than 18 hours.

A series of small-scale anion exchange chromatographic steps can beemployed to optimize separation of the sought-after antiangiogenicactivity generated in plasma by the tPA and captopril treatment from thebulk of the plasma proteins. We found that the activity had a fairlystrong affinity to the anion exchange resins. Therefore, plasma wasfirst exchanged into buffer A (10 mM Tris HCl pH 7.4)/300 mM NaCl andloaded onto a HiTrap QXL column. Three fractions are collected whenproteins are eluted with 300 (flow through), 400, and 1000 mM NaCl. Onlythe 400-mM NaCl fraction (FIG. 9, lane 3) contains a potent anti-tubeformation activity (FIG. 10D), whereas the flow through (FIG. 9, lane 2)and the 1000 mM NaCl wash (FIG. 9, lane 4) have little or no activity(FIGS. 10C and 10E). Although the treated plasma (FIG. 9, lane 1)contains significant anti-tube formation activity (FIG. 10B), theactivity present in the 400 mM NaCl fraction appears to be more potentthan the input treated plasma (FIGS. 10B and 10D).

The active fraction from the anion exchange column is furtherfractionated on a HiTrap Blue-Sepharose column. We found that theantiangiogenic activity could best be purified on this column by athree-step-elution method. The sample is first loaded at 200 mM NaCl,followed by an extensive wash with the loading buffer to yield aflow-through fraction (FIG. 9, lane 5) that has no effects onendothelial cell tube formation in vitro (FIG. 10F). Using thisoptimized loading condition, the antiangiogenic activity is completelyretained on the column and can be recovered by an elution with 1.5 MNaCl (FIG. 9, lane 6 and FIG. 10G). Tightly bound material released by a2M guanidine hydrochloride wash (FIG. 9, lane 7) has littleantiangiogenic activities (FIG. 10H) as determined by our tube formationassay.

The final step of the purification scheme involves using immobilizedmetal affinity chromatography (IMAC). This technique is particularlyuseful in separating proteins of mammalian origin due to theirrelatively high cysteine and histidine contents. Successful employmentof IMAC depends on various factors, including the metal chosen, thestructure of the chelators residing on the resins, and the loadingconditions. Our pilot experiments show that the Ni/NTA combination givesthe highest selectivity when the input proteins are loaded in 0.8 Msodium sulfate/50 mM sodium phosphate pH 7.0. The 1.5 M NaCl fractionfrom the Blue-Sepharose column is first exchanged into the loadingbuffer by a NAP-10 column and loaded onto a Ni-NTA Superflow column.Antiangiogenic protein passes to the flow through fraction (FIG. 9 lane8 and FIG. 10I). Proteins bound to the resins are eluted with 200 mMimidazole (FIG. 9, lane 9) and show little antiangiogenic effects (FIG.10J).

Under these optimized conditions, the antiangiogenic moiety in thetreated plasma is purified to near homogeneity (>95% pure). SDS-PAGEreveals the active moiety appears to have a molecular weight between 70to 90 kDa (FIG. 9, lane 8). This band was excised and submitted forprotein sequencing by MALDI-TOF. Comparison of the mass spectral data ofthe protein fragments to a database of known protein fragmentsidentified prothrombin as the candidate.

Prothrombin is antiangiogenic. To examine whether the prothrombinpurified from tPA/captopril-treated plasma was altered, a protein samplewas analyzed by mass spectroscopy. The data revealed that theprothrombin purified from tPA/captopril-treated plasma had a molecularweight of 71904.7, similar to that of a commercially available sample ofprothrombin (71441). This suggests that prothrombin was most likely notaltered proteolytically by the tPA/captopril treatment. The minordifference in the molecular weights apparently was not related toactivities, because the commercially available prothrombin alsodisrupted endothelial cell tube formation (FIGS. 11B and 11C) andappeared to have a similar potency as the prothrombin purified from thetreated plasma. Collectively, our data suggest that prothrombin isantiangiogenic. Although normally suppressed in plasma, this activity ofprothrombin is unmasked by treatment with tPA/captopril.

Protease activity of thrombin is antiangiogenic. Since prothrombin isthe inactive precursor of thrombin, we carried out a series ofexperiments to determine whether thrombin exhibits an antiangiogenicactivity similar to prothrombin. Thrombin appears to have minimaleffects on endothelial cell tube formation in vitro when used at 5 U/ml(FIGS. 11D and 11E). However, thrombin at 10 U/ml significantly inhibitstube formation (FIG. 11F). The inhibition by either prothrombin orthrombin was completely blocked by the addition of lepirudin, a specificinhibitor of thrombin (FIGS. 11G and 11H). This suggests that bothprothrombin and thrombin mediates the antiangiogenic behavior throughthe protease activity of thrombin. Most importantly, in the presence of200 U/ml lepirudin, endothelial cells appear to form tubes with similarefficiency when incubated with either tPA/captopril-treated (FIG. 11J)or untreated plasma (FIG. 11J). Lepirudin at 200 U/ml apparently had noeffects in endothelial cell tube formation (FIG. 11K). Therefore, themajority of the induced activities in plasma were due to the thrombin'sproteolytic property.

Role of PARs. Thrombin signals through a class of cell surface receptorknown as the protease-activated receptors (PARs). Four PARs have beenidentified to date. PAR-1, 2, and 3 are expressed on human endothelialcells. Amongst these three receptors, thrombin specifically activatesPAR-1 and PAR-3. The manner by which thrombin activates these receptorsis novel; thrombin binds and cleaves the receptor. The newly exposedN-terminus of the receptor acts as its own ligand and activates thereceptor. This leads to the development of peptides corresponding to thenewly exposed N-terminal amino acid sequence of the activated receptorsto activate PARs specifically.

Therefore, we asked whether addition of a peptide corresponding to theN-terminal 16 amino acids of activated PAR-1 (the TRAP peptide) toendothelial cells would recapitulate thrombin's antiangiogenic effects.Indeed, TRAP dose-dependently inhibited endothelial cell tube formationin vitro (FIG. 12). This observation strongly suggests that activationof PAR-1 is sufficient to inhibit the ability of endothelial cells toform tubes in vitro. in vivo angiogenesis assays. To ascertain the invivo relevance of this novel activity, we performed matrigel-plug assaysin mice. Protirombin, TRAP, or PBS (carrier) was mixed in matrigelcontaining bFGF and injected subcutaneously into mice. ten days afterthe injections, the matrigel plugs were excised, sectioned, and stainedwith H&E. The number of blood vessels formed was counted and used as anindication of neovascularization. In the absence of a stimulus, minimalamount of red-blood-cell-containing micro-vessels were seen (FIG. 13A).As expected, bFGF effectively stimulated blood vessel formation in theimplanted matrigel plugs (FIG. 13B). Approximately a seven-fold increasein angiogenesis was observed (FIG. 13E). However, this stimulation wassignificantly reduced by both prothrombin (FIG. 13C) and TRAP (FIG. 13D)in this in vivo angiogenesis model in a dose-dependent manner (FIG.13E), consistent with the results obtained from our in vitroexperiments.

Next, we examined the effects of activating different PARs by smallpeptides on endothelial cell tube formation. These peptides are specificactivators of either PAR-1 (SFLLRN), 2 (SLIGKV), or 4 (GYPGKF).Additionally, a mutant of the PAR-1 activating peptide (SALLRN) was alsoincluded as a control. A single amino acid mutation from phenylalanineto alanine at position 2 completely eliminated the ability of thepeptide to induce platelet aggregation in vitro. Contribution of PAR-3was not tested, because no PAR-3 activating peptide has been describedso far. Activation of PAR-2 or PAR-4 on endothelial cells using thespecific peptides at the concentrations tested, bore little effect ontube formation (FIGS. 14E and 14F). However, SFLLRN showed adose-dependent inhibitory effect on endothelial cell tube formation(FIGS. 14B and 14C), consistent with the results obtained by using TRAPpeptide. In contrast, the mutant peptide SALLRN showed no inhibitoryeffect when used at the same concentration (FIG. 14D). Collectively,these observations suggest that activation of PAR-1 inhibited HUVECs toform tubes on matrigel.

Experimental Procedures

Materials. TRAP (SFLLRNPNDKYEPF) was obtained from Sigma-Aldrich (St.Louis, Mo.). Four short peptides (SFLLRN, SALLRN, GYPGKF, and SLIGKV)were synthesized as C-terminal amides and were purified by high-pressureliquid chromatography. Prothrombin and thrombin were purchased fromCalbiochem (San Diego, Calif.). Matrigel was obtained from BDBiosciences (Bedford, Mass.). Lepirudin and tPA were purchased fromAventis Pharmaceutical (Kansas City, Mo.) and Genentech (San Francisco,Calif.), respectively. bFGF was purchased from Peprotech (Rocky Hill,N.J.), and captopril was obtained from Sigma-Aldrich Research. Cells.Human umbilical vein endothelial cells (HUVECs) and EGM2-MV medium werepurchased from BioWhittaker (Walkersville, Md.). Cells were culturedaccording to supplier's instructions.

Purification. A series of small-scale anion exchange chromatographicsteps was initially employed to optimize separation of theantiangiogenic activities generated in plasma by the tPA and captopriltreatment from the bulk proteins. Treated plasma (1 ml) was exchangedinto buffer A (10 mM Tris HCl pH 7.4)/50 mM NaCl by a NAP-10 column(Pharmacia, Piscataway, N.J.). The sample was applied onto a 1-ml HiTrapQXL (Pharmacia) pre-equilibrated with buffer A/50 mM NaCl at 1 ml/min.The column was washed with the start buffer until the absorbance at 280nm returned to baseline. Proteins were eluted by a step gradient of NaCl(50-mM increments) until 500 mM NaCl was reached. The column was thenwashed with buffer A/1 M NaCl. All fractions were concentrated andexchanged with 1×PBS before testing for activities. The antiangiogenicactivities eluted between 300 and 400 mM NaCl.

We performed preparative-scale separation of protein fractions byapplying treated plasma onto a 20-ml HiPrep 16/10 Q XL column(Pharmacia). The column was washed extensively with Buffer A/300 mMNaCl. Absorbed proteins were eluted from the column sequentially withbuffer A/400 mM NaCl and buffer A/1 M NaCl. All fractions wereconcentrated and exchanged with 1×PBS and stored at −20° C. The activefraction of the Q-Sepharose column was then exchanged into 50 mM sodiumphosphate pH 7.0/200 mM NaCl by NAP-10 columns and loaded onto a 5-mlHiTrap Blue Sepharose column (Pharmacia) pre-equilibrated with theloading buffer. After extensive washing, the antiangiogenic activity waseluted with 50 mM sodium phosphate pH 7.0/1.5 M NaCl. Irreversibly boundproteins were stripped off the column using 2 M guanidine hydrochloride.All fractions were concentrated and exchanged into 1×PBS and stored at−20° C. The active fraction from the HiTrap Blue column was exchangedinto 0.8 M sodium sulfate/50 mM sodium phosphate by using a NAP 10column and loaded onto a 5-ml Ni-NTA Superflow (Qiagen; Valencia,Calif.) column pre-equilibrated with the loading buffer. The column waswashed extensively, following by an elution with 200 mM imidazole/50 mMsodium phosphate pH 7.0. All fractions were concentrated, exchanged into1×PBS, and stored at −20° C.

Protein sequencing and molecular weight determination. The purifiedprotein was subjected to protein sequencing by mass spectroscopy. Massspectral data of the protein fragments were compared to the databaseNCBInr 200001111 using the search engine Mascot. This analysis revealedprothrombin as the candidate with a Mouse Score of 137. To determinemolecular weights by mass spectroscopy, protein samples were furthercharacterized using standard techniques.

In vitro treatment of human plasma. In vitro treatment of plasma withtPA and captopril may be performed. Briefly, 1 ml of plasma is incubatedwith 10 μg/ml tPA and 1 μM captopril at 37° C. for three hours. Samplescan be stored at −20° C. until use.

Matrigel tube formation assays. Samples collected from the purificationprocess were exchanged into 1×PBS and reconstituted into 1×concentration (by volume) compared to the input tPA/captopril-treatedplasma. They were tested in the matrigel tube formation assays at 10%(v/v) as follows. Unpolymerized matrigel (7 mg/ml) was placed in thewells (100 μl/well) of a pre-chilled 48-well cell culture plate and thenincubated at 37° C. for 30-45 minutes for polymerization to take place.HUVECs (4×10⁴ in 300 μl of EGM2-MV with 5% fetal bovine serum (FBS),gentamicin sulfate, amphotericin B, hydrocortisone, ascorbic acid, VEGF,bFGF, hEGF, and R³-IGF-1) were treated with the agent tested, platedonto the matrigel-coated plates, and incubated at 37° C. for 12-16hours. Tube formation was examined (4× magnification) through aninverted phase contrast microscope (Nikon Corporation; Tokyo, Japan) andrecorded by a Spot RT camera (Diagnostic Instruments Inc; SterlingHeights, Mich.) using an automated image capture software (Compix IncImaging Systems; Township, Pa.).

In vivo matrigel-plug assays. The matrigel-plug assay was performed asdescribed in Maeshima et al., (Maeshima et al., J Biol Chem 275:21340-8,2000; Maeshima et al., J Biol Chem 276:31959-68, 2001) withmodifications as follows. Briefly, 500 μl of unpolymerized matrigel at aconcentration of 10 mg/ml enriched with bFGF (250 ng/ml) were mixed withone of the following: PBS (vehicle), prothrombin, or TRAP. Each mixturewas injected subcutaneously at the left lower abdominal wall of C57/Bmice (5 to 6-week old; Jackson laboratories; Bar Harbor, Me.). At dayten, the mice were sacrificed. The matrigel plugs were then excised,fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and H&Estained. Sections were examined by light microscopy, and the totalnumber of blood vessels from 10 high power fields (400×) were counted ina blinded fashion. Only the micro-vessels containing red blood cellswere counted as positive. Results shown represent the average of countsfrom 4 to 5 matrigel plugs per group.

EXAMPLE 3 Identification of Compounds that Modulate uPA-uPA-R BiologicalActivity

There has been controversy in the art regarding the effects of proteasesin tumor growth and progression. Based upon our results, we believe thatoverexpression of proteases of the plasminogen activator family (e.g.,urokinase and tissue plasminogen activator) by the tumor may prevent ordelay tumor growth, metastases, and improve survival.

While not wishing to be bound by a particular mechanism, we believethese proteases may degrade fibrin, known to occur in the matrix of manytumors and which is known to favor angiogenesis and support tumor cellgrowth. The goals of the experiments performed were to determine theeffects of tumor overexpression of uPA, tPA and a mutant form of uPA,which does not bind to its receptor, but preserves the proteolyticactivity.

Construction of mutant uPA clones and cell transfection. We chose topursue a genetic approach, in which we stably transfected an aggressiveand highly metastatic murine breast cancer cell line (4T1) with thegenes for wild type uPA, tPA and a receptor binding mutant of uPA(uPAm). We inserted the cDNA encoding murine tPA into pcDNA 3.1 (+)(Invitrogen), a CMV driven mammalian expression vector, by restrictionenzyme digestion of BamHI and NheI. Wild type uPA cDNA was inserted tothe pcDNA 3.1 (+) expression vector and the transfected into 4T1 cellsin a similar fashion as described for the tPA cDNA. Residues 22, 27, 29and 30 on the growth factor domain of the murine uPA molecule have beenimplicated in binding to mouse uPAR.

Indeed, for mouse uPA, a triple mutation at residues 27, 29 and 30 hasbeen shown to abrogate uPA binding to its receptor. We introduced thesemutations by multi-site-directed mutagenesis using a clone of wild typeUPA generated by RT-PCR of the murine cell line Lewis lung carcinomaRNA.

The oligonucleotides used to create the triple mutation changing Arg 27,29, 30 into Asn 27, His 29, Trp 30 were as follows: (forward primer) 5′P-CCT ACA AGT ACT TCT CCA ACA TTC ACT GGT GCA GCT GCC CAA GG 3′ and(reverse) 5′ P-CCT TGG GCA GCT GCA CCA GTG AAT GTT GGA GAA GTA CTT GTAGG 3′. The resulting mutant will have lost an EcoRI site at position143, which was used to initially screen different mutant clonesgenerated. After this, we inserted the mutant uPA cDNA into the pcDNA3.1 (+) vector. Prior to introducing the vector constructs into thecancer cell lines, we sequenced the constructs and performed in vitrotranscription-translation assays, in order to confirm that the DNAproducts were correct and that they transcribed.

The pcDNA 3.1-tPA, pcDNA 3.1-wild type uPA, pcDNA 3.1-uPAm constructs,as well as an empty vector control were introduced into 4T1 cells bytransfection using lipofectamine (Gibco-BRL) reagent. We selected 12-14stable single clones in each group with 500 ug/mL of hygromycin. Each ofthe clones was evaluated for generation of the gene of interest bynorthern blot. The clones that had the highest generation of the genesof interest were selected. These were: tPA clone 9, wild type uPA clone11, and uPA mutant clone 11. Pools of stable clones of pcDNA 3.1-tPA andpcDNA 3.1 (+) were also selected using 500 ug/mL of hygromycin.

Experiments using transfected tumor cells. Tumor cells (10⁵ cells in 50uL) were injected into the left 5th mammary pad of female (4-6) week oldBALB/c mice (Charles River Labs, Cambridge, Mass.). Groups of 12 animalswere injected with cells transfected with empty vector and cellstransfected with tPA cDNA, wild type uPA cDNA, and mutant uPA cDNA.Primary tumor size was measured every other day for a total of fiveweeks using calipers and calculated using the standard formula(width2×length×0.52). FIG. 15 demonstrates the effects of tissueproteases in a murine breast cancer model. Results demonstrate primarytumor cells expressing tissue proteases are more resistant to tumorgrowth, relative to hygromycin-resistant 4T1 control cells, and thatexpression of mutants, defective for uPA receptor binding, is mosteffective in inhibiting tumor growth. Expression of wildtype uPA and tPAalso significantly inhibit tumor cell growth, albeit less effectively.At 5 weeks post tumor cell implantation, animals were euthanized; andthe primary tumors and lungs were removed and sectioned.

High throughput assays and screens. Other methods of observing changesto uPA-uPA-R interactions and subsequent biological activity may beexploited in high throughput assays for the purpose of identifyingcompounds that modulate this protein-protein interaction. Compounds thatinhibit uPA from binding to uPA-R without affecting uPA proteolyticactivity may be identified by such assays. Such identified compounds mayhave utility as therapeutic agents in the treatment of angiogenicdisorders.

There are many methods known in the art to screen for modulators ofprotein-protein interactions and they are applicable here. One method isto immobilize one component (for example, the uPA-receptor) to a solidsupport matrix. The second component (for example, urokinase) is labeled(either through the use of radioactive isotopes such as ³²P or ³⁵S, orthrough non-radioactive alternatives such as fluorophores) and allowedto form a complex with the immobilized receptor. This complex is readyfor screening with candidate compounds. A compound that displaces uPAfrom the uPA- receptor can be readily measured by release ofradioactivity or fluorescence.

Alternatively, compounds can be contacted to immobilized uPA-R, followedby addition of the labeled uPA polypeptide. Compounds that modulateuPA-uPA-R interactions could also be measured by release of eitherradioactivity or fluorescence.

Therapeutic Uses

The invention features methods for treating angiogenesis associateddiseases or disorders by administering polypeptide or nucleic acidcompounds. Compounds of the present invention may be administered by anyappropriate route for treatment or prevention of a disease or conditionassociated with angiogenesis associated diseases. These may beadministered to humans, domestic pets, livestock, or other animals witha pharmaceutically acceptable diluent, carrier, or excipient, in unitdosage form. Administration may be parenteral, intravenous,intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital,ophthalmic, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intranasal, aerosol, by suppositories,or oral administration.

Therapeutic formulations may be in the form of liquid solutions orsuspensions; for oral administration, formulations may be in the form oftablets or capsules; and for intranasal formulations, in the form ofpowders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins).Formulations for parenteral administration may, for example, containexcipients, sterile water, or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate formulations (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Formulationsfor inhalation may contain excipients, for example, lactose, or may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycholate and deoxycholate, or may be oily solutions foradministration in the form of nasal drops, or as a gel. Theconcentration of the compound in the formulation will vary dependingupon a number of factors, including the dosage of the drug to beadministered, and the route of administration.

The compound may be optionally administered as a pharmaceuticallyacceptable salt, such as a non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like.

Administration of compounds in controlled release formulations is usefulwhere the compound of formula I has (i) a narrow therapeutic index(e.g., the difference between the plasma concentration leading toharmful side effects or toxic reactions and the plasma concentrationleading to a therapeutic effect is small; generally, the therapeuticindex, TI, is defined as the ratio of median lethal dose (LD₅₀) tomedian effective dose (ED₅₀)); (ii) a narrow absorption window in thegastro-intestinal tract; or (iii) a short biological half-life, so thatfrequent dosing during a day is required in order to sustain the plasmalevel at a therapeutic level.

Many strategies can be pursued to obtain controlled release in which therate of release outweighs the rate of metabolism of the therapeuticcompound. For example, controlled release can be obtained by theappropriate selection of formulation parameters and ingredients,including, eg., appropriate controlled release compositions andcoatings. Examples include single or multiple unit tablet or capsulecompositions, oil solutions, suspensions, emulsions, microcapsules,microspheres, nanoparticles, patches, and liposomes.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andantiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc).

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent, or as soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium.

Administration of tissue proteases. By selectively disrupting orpreventing tissue proteases, such as tPA and uPA, from binding to theirnatural receptor(s) the polypeptides of the invention, or derivatives orpeptidomimetics thereof, can significantly decrease angiogenic potentialresulting in reduction or ablation of neoplastic cell survival orgrowth. Therefore, the polypeptides of the invention, or derivatives orpeptidomimetics thereof, can be used in the treatment of cancer or otherneoplasms or even other angiogenesis-associated diseases (e.g., maculardegeneration of the eye).

Angiogenesis-associated disorders include, cancer, rheumatoid arthritis,psoriasis, pyogenic granuloma, diabetic retinopathy, maculardegeneration, corneal graft neovascularization, hypertrophic scarring,angiofibroma, Osler-Weber syndrome, neovascular glaucoma, andscleroderma.

Cancers and other neoplasms include, without limitation, leukemias(e.g., acute leukemia, acute lymphocytic leukemia, acute myelocyticleukemia, acute myeloblastic leukemia, acute promyelocytic leukemia,acute myelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease,non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chaindisease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma).

For any of the methods of application described above, the tissueprotease, fragment or mutant thereof, or peptidomimetic small moleculemay be applied to the site of the needed therapeutic event (for example,by injection), or to tissue in the vicinity of the predicted therapeuticevent or to a blood vessel supplying the cells predicted to requireenhanced therapy.

The dosage of a tissue protease, fragment or mutant thereof, orpeptidomimetic small molecule depends on a number of factors, includingthe size and health of the individual patient, but, generally, between0.1 mg and 100 mg/kg body weight, is administered per day to an adult inany pharmaceutically acceptable formulation. In addition, treatment byany of the approaches described herein may be combined with moretraditional therapies.

Combination therapy. If desired, polypeptides of the invention may beadministered alone or in combination with a second, third, fourth, oreven fifth therapeutic agent. Combination therapy may be performed aloneor in conjunction with another therapy (e.g., surgery, y-radiation,chemotherapy, biologic therapy). Additionally, a person having a greaterrisk of developing a neoplasm (e.g., one who is genetically predisposedor one who previously had a neoplasm) may receive prophylactic treatmentto inhibit or delay neoplastic formation. The duration of thecombination therapy depends on the type of disease or disorder beingtreated, the age and condition of the patient, the stage and type of thepatient's disease, and how the patient responds to the treatment.

The dosage, frequency and mode of administration of each component ofthe combination can be controlled independently. For example, onecompound (i.e., the tissue protease) may be administered intravenouslyonce per day, while the second compound (i.e., the antiproliferative)may be administered orally twice per day. Combination therapy may begiven in on-and-off cycles that include rest periods so that thepatient's body has a chance to recovery from any as yet unforeseenside-effects. The compounds may also be formulated together such thatone administration delivers both compounds.

Exemplary antiproliferative agents, include alkylating agents (e.g.,nitrogen mustards such as cyclophosphamide, ifosfamide, trofosfamide,and chlorambucil; nitrosoureas such as carmustine, and lomustine;alkylsulphonates such as bisulfan and treosulfan; triazenes such asdacarbazine; platinum-containing compounds such as cisplatin andcarboplatin), plant alkaloids (e.g., vincristine, vinblastine,anhydrovinblastine, vindesine, vinorelbine, paclitaxel, and docetaxol),DNA topoisomerase inhibitors (e.g., etoposide, teniposide, topotecan,9-aminocamptothecin (campto), irinotecan, and crisnatol), mytomycins(e.g., mytomicin C), antifolates (e.g., methotrexate, trimetrexate,mycophenolic acid, tiazofurin, ribavirin, EICAR, hydroxyurea, anddeferoxamine), uracil analogs (5-fluorouracil, floxuridine,doxifluridine, and ratitrexed), cytosine analogs (cytarbine, cytosinearabinoside, and fludarabine), purine analogs (e.g., mercaptopurine, andthioguanine), hormonal therapies (e.g., tamoxifen, raloxifene,megestrol, goserelin, leuprolide acetate, flutamide, and bicalutamide),vitamin D3 analogs (EB 1089, CB 1093, and KH 1060), vertoporfin,phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A,interferon-α, interferon-γ, tumor necrosis factor, lovastatin,1-methyl-4-phenylpyridinium ion, staurosporine, actinomycin D, bleomycinA2, bleomycin B2, adriamycin, peplomycin, daunorubican, idarubican,epirubican, pirarubican, zorubican, mitoxantrone, verapamil, and the uPAoctamer-capped peptide, A6.

Gene therapy. Gene therapy is another potential therapeutic approach inwhich nucleic acids encoding tissue proteases such as tPA and uPA, whichare incapable of binding to their cognate receptor, are introduced intocells. The gene must be delivered to those cells in a form in which itcan be taken up and encode for sufficient protein to provide effectivefunction.

Transducing retroviral, adenoviral, and human immunodeficiency viral(HIV) vectors can be used for somatic cell gene therapy especiallybecause of their high efficiency of infection and stable integration andexpression (see, for example, Cayouette and Gravel, Hum. Gene Ther.,8:423-430, 1997; Kido et al. Curr. Eye Res., 15:833-844, 1996; Bloomeret al., J. Virol., 71:6641-6649, 1997; Naldini et al., Science272:263-267, 1996; Miyoshi et al., Proc. Natl. Acad. Sci. USA,94:10319-10323, 1997). For example, uPA nucleic acid, or portionsthereof, can be cloned into a retroviral vector and driven from itsendogenous promoter or from the retroviral long terminal repeat or froma promoter specific for the target cell type of interest (such asendothelial cells). Other viral vectors, which can be used, includeadenovirus, adeno-associated virus, vaccinia virus, bovine papillomavirus, vesicular stomatitus virus, or a herpes virus such asEpstein-Barr Virus.

Gene transfer could also be achieved using non-viral means requiringinfection in vitro. This would include calcium phosphate, DEAE-dextran,electroporation, and protoplast fusion. Liposomes may also bepotentially beneficial for delivery of DNA into a cell. Although thesemethods are available, many of these are of lower efficiency.

Production of tissue protease containing vectors. Tissue proteases canbe produced by any method known in the art for the expression ofrecombinant proteins. Nucleic acids that encode tissue proteases may beintroduced into various cell types or cell-free systems for expressionthereby allowing small-, large-, and commercial-scale production,purification, and patient therapy.

Eukaryotic and prokaryotic tissue protease expression systems may begenerated in which a tissue protease-coding sequence is introduced intoa plasmid or other vector, which is then used to transform living cells.Constructs in which the tissue protease cDNA contains the entire openreading frame or biologically active fragment thereof, are inserted inthe correct orientation into an expression plasmid and may be used forprotein expression. It is understood that the expression of tissueproteases in eukaryotic expression systems has the added benefit ofbeing post-translationally processed in the appropriate cellularorganelle(s). Secreted proteins can be processed by proteolyticprocessing by proteases residing at the extracellular face of the cell,such as the proprotein convertases (PCs). Optionally, production oftissue proteases, such as tPA and uPA can be attained by fusing thecorresponding nucleic acid sequence immediately following an initiatormethionine (AUG). Translation of the resulting mRNA in any prokaryoticor eukaryotic host would lead to the cleavage of the initiatormethionine by a methionine aminopeptidase (MetAP). MetAPs have beenextensively studied and have been shown to cleave the initiatormethionine residue if the amino acid at position 2 (i.e., following themethionine) is glycine, alanine, serine, threonine, proline, cysteine,or valine (Arfine et al., Proc. Natl. Acad. Sci. USA, 92:7714-7718,1995; Bradshaw et al., Trends Biochem. Sci., 23:263-267, 1998; Lowtherand Matthews, Biochim. Biophys. Acta, 1477:157-167, 2000).

Tissue proteases can be expressed as soluble cytoplasmic proteins, orpreferably, fused in-frame with a secretory signal peptide to beexpressed as secreted recombinant polypeptides. Preferably, thesecretory signal is based on either the A or α-factor secretory signalof Saccaromyces cereviseae. Prokaryotic and eukaryotic expressionsystems also allow for the expression and recovery of tissue proteasefusion proteins in which the tissue protease is covalently linked to atag molecule on either the amino terminal or carboxy terminal side,which facilitates identification and/or purification. Examples of tagsthat can be used include hexahistidine, HA, FLAG, and c-myc epitopes.Larger fusion tags may also be used and includeglutathione-S-transferase, maltose binding protein, cellulose bindingprotein, and protein-A. An enzymatic or chemical cleavage site can beengineered between the tissue protease and the tag moiety so that thetag can be removed following purification.

If desired, the tissue protease may also be engineered as a fusionprotein containing one member of a binding pair to facilitate proteinpurification. Exemplary binding pairs include without limitationantigen-antibody, biotin-avidin or biotin-strepavidin, hormone-hormonereceptor, receptor-ligand, enzyme-substrate, IgG-protein A, andGST-glutathione.

Host Cells. Once a tissue protease expression vector is constructed, itis introduced into an appropriate host cell by transformationtechniques, such as, but not limited to, calcium phosphate transfection,DEAE-dextran transfection, electroporation, bombardment, microinjection,protoplast fusion, dendrimer-mediated transfection, or liposome-mediatedtransfection. The host cells that are transfected with the vectors ofthis invention may include (but are not limited to) E. coli or otherbacteria, yeast, fungi, insect cells (using, for example, baculoviralvectors for expression in Sf9 or Sf21 insect cells), or cells derivedfrom murine, human, or other animals. Those skilled in the art ofmolecular biology will understand that a wide variety of expressionsystems and purification systems may be used to produce recombinanttrefoil peptides and mixtures thereof. Suitable host cells include, forexample, yeast, bacteria, insect cells, mammalian cells. Desirable yeastcells include Saccaromyces cereviseae, Schizosaccaromyces pombe, or themethylotrophic yeast, Pichia pastoris. Insect cells include Sf9 cells,Sf21, and Schneider cells. Mammalian cells include NIH-3T3, C3H10T1/2,HeLa, HEK293, COS, CV, and CHO cells.

Alternatively, bacterial host cells, such as E. coli may be used. Inaddition to E. coli, other bacterial species are also useful topropagate and/or express tissue proteases in a manner similar to usingE. coli. For instance, Lactobacilli and Bifidobacterium species may beused to express the tissue protease either as soluble cytoplasmicproteins or by creating chimeric fusion proteins in which signalpeptides would direct the expressed proteins into the periplasmicregions, to the outer surface of the bacteria, or as a secreted productout of the cell. Both Lactobacilli and Bifidobacterium spp can befurther utilized to express foreign proteins in the preparation ofconsumable food products, for example, in making yogurt or other dairyproducts.

Protein Purification. Once a recombinant protein is expressed, it can beisolated from cell lysates if expressed as a cytoplasmic protein, orfrom the media if expressed as a secreted protein. Recombinant chimericproteins bearing the A or α-factor secretory signal in yeast expressionsystems, for example, are exported out of the cell and can be collectedfrom the culture media for further purification (see, for example, U.S.Pat. Nos. 4,808,537, 4,837,148, 4,879,231, 4,882,279, 4,818,700,4,895,800, and 4,812,405, 5,032,516, 5,122,465, 5,268,273; herebyincorporated by reference).

Protein purification techniques such as ion-exchange, gel-filtration,and affinity chromatography can be utilized to isolate intestinaltrefoil peptides from unwanted cellular proteins. Once isolated, therecombinant protein can, if desired, be purified further by highperformance liquid chromatography (HPLC; e.g., see Fisher, LaboratoryTechniques In Biochemistry And Molecular Biology, Work and Burdon, Eds.,Elsevier, 1980).

If the tissue protease is fused in frame with a binding pair member, the25 tissue protease can be isolated using a purification method based onthe binding interaction. For example, a tissue protease fusioncontaining a biotin acceptor domain may be expressed in the yeast Pichiapastoris. Following in vitro biotinylation with biotin ligase andsolubilization of crude membrane fractions with a detergent, the tissueprotease can be purified using a combination of chromatographictechniques. Such a system is described in detail by Julien et al.(Biochemistry 39:75-85, 2000). Alternatively, if large-scale productionof tissue proteases is required, any protein purification method knownin the art may be used. An exemplary method is aqueous two phase systemsand is described by Cunha T., et al (Mol. Biotechnol, 20: 29-40, 2002).

Protein modifications. It is desirable that tissue proteases of theinvention lack receptor binding activity. For example, it is known thatamino acids 24-30 (the Ω-loop) of uPA is important for receptorassociation. Substitutions at any of these residues, or residuesresiding outside this loop and identified to regulate binding of uPA toits receptor, would be of benefit. In a desirable embodiment, amino acidsubstitutions are directed to the sequence²⁴tyr-²⁵phe-²⁶ser-²⁷asn-²⁸ile-²⁹his-³⁰trp in human,²⁴tyr-²⁵phe-²⁶ser-²⁷arg-²⁸ile-²⁹arg-³⁰arg in mouse, and²⁴tyr-²⁵phe-²⁶ser-²⁷ser-²⁸ile-²⁹arg-³⁰arg in rat. In another desirableembodiment, any 2, 3, 4, 5, 6, or all 7 amino acids of uPA may besubstituted with another amino acid, typically a non-conservative aminoacid. Amino acid residues in the Ω-loop may be substituted from onespecies to another. For example, a triple mutant of murine uPAincorporating the human amino acid residue substitutions at positions27, 29, and 30 (i.e., R27N, R29H, and R30W) has been shown to ablatebinding of murine uPA to the mouse UPA-R receptor.

Receptor binding mutants of uPA may further possess mutations ormodifications modulating biological activity, for example, increased,decreased, or ablated catalytic activity, mutations affecting proteinphosphorylation (e.g., Ser138), and mutations affecting substratespecificity.

Advanced protein engineering technologies can be incorporated to develophuman protein pharmaceuticals with enhanced therapeutic properties. Mostprotein pharmaceuticals are rapidly eliminated by the body, which limitstheir effectiveness and requires that they be administered by frequent,often daily, injection. The most commonly employed method for extendingprotein half-life is PEGylation. PEGylating proteins uses compounds suchas N-hydroxysuccinimide (NHS)-PEG to attach PEG to free amines,typically at lysine residues or at the N-terminal amino acid. The PEGmoiety attaches to the protein randomly at any of the available freeamines, resulting in a heterogeneous product mixture consisting ofmono-, di-, tri-, etc., PEGylated species modified at different lysineresidues.

Site-specific PEGylation may be employed. Site-Specific PEGylationallows a protein to be selectively modified with PEG at a single,unique, pre-determined site. The site of PEGylation potentially can beany amino acid position in the protein and can be varied depending uponthe protein. By targeting the PEG molecule to an optimal site in aprotein, it is possible to create PEGylated proteins that arehomogeneously modified and have no significant loss of biologicalactivity.

An alternative technology takes advantage of the modular structure andlong circulating half-lives of human immunoglobulins (antibodies).Recombinant DNA methods are employed to covalently fuse therapeuticproteins to the Fc domains of human immunoglobulin gamma proteins(IgGs). IgGs are abundant proteins that have circulating half-lives ofup to 21 days in humans. In a desirable embodiment, human or humanizedimmunoglobins are used as a fusion. It is contemplated that theimmunoglobins may also be PEGylated and glycosylated to further increasehalf-life, or decrease immune detection.

Fusion tags or post-translational modifications can be used with thetissue proteases. For example, the tissue proteases can beasialo-glycosylated. It is known that liver cells express theasialo-glycoprotein receptor. Asialo-glycosylated proteins would thusaccumulate in the liver, increasing therapeutic bioavailability inpathologies affecting the liver. Analogous fusion tags and/ormodifications can be incorporated to the polypeptides of the inventiondirecting them to specific organs or tissues. For example, fusion tagscan be made with non-functional EGF-Rc-binding epidermal growth factor;many breast cancers have been found to over-express the EGF receptor anda non-functional epidermal growth factor would be able to target thetissue proteases to the cancerous cells.

Test extracts and compounds. In general, compounds that affect PAR oruPA/uPA-R receptor signaling are identified from large libraries of bothnatural products, synthetic (or semi-synthetic) extracts or chemicallibraries, according to methods known in the art.

Those skilled in the art will understand that the precise source of testextracts or compounds is not critical to the screening procedure(s) ofthe invention. Accordingly, virtually any number of chemical extracts orcompounds can be screened using the exemplary methods described herein.Examples of such extracts or compounds include, but are not limited to,plant-, fungal-, prokaryotic- or animal-based extracts, fermentationbroths, and synthetic compounds, as well as modifications of existingcompounds. Numerous methods are also available for generating random ordirected synthesis (e.g., semi-synthesis or total synthesis) of anynumber of chemical compounds, including, but not limited to,saccharide-, lipid-, peptide-, and nucleic acid-based compounds.Synthetic compound libraries are commercially available from, forexample, Brandon Associates (Merrimack, N.H.) and Aldrich Chemical(Milwaukee, Wis.).

Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant, and animal extracts are commercially available from anumber of sources, including, but not limited to, Biotics (Sussex, UK),Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce,Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, naturaland synthetically produced libraries are produced, if desired, accordingto methods known in the art (e.g., by combinatorial chemistry methods orstandard extraction and fractionation methods). Furthermore, if desired,any library or compound may be readily modified using standard chemical,physical, or biochemical methods.

Other Embodiments. From the foregoing description, it is apparent thatvariations and modifications may be made to the invention describedherein to adopt it to various usages and conditions. Such embodimentsare also within the scope of the following claims.

All publications mentioned in this specification are herein incorporatedby reference to the same extent as if each independent publication orpatent application was specifically and individually indicated to beincorporated by reference.

1. A method for the treatment of angiogenesis-associated diseases, saidmethod comprising administering a therapeutic amount of a pharmaceuticalcomposition comprising a Protease-Activated Receptor (PAR) agonist,wherein said agonist is capable of binding directly to the PAR receptor.2. A method for the treatment of angiogenesis-associated diseases, saidmethod comprising administering a therapeutic amount of a compound whichresults in activation of a Protease-Activated Receptor (PAR), whereinsaid treatment does not comprise administering either tissue plasminogenactivator (tPA) polypeptide or a urokinase plasminogen activator (uPA),wherein said uPA is capable of binding to the human uPA receptor (uPA-R)in combination with captopril.
 3. A pharmaceutical compositioncomprising (i) substantially pure PAR-agonist, wherein said agonist iscapable of binding directly to the PAR receptor; and (ii) apharmaceutically acceptable carrier.
 4. A pharmaceutical compositioncomprising (i) a therapeutic amount of a compound which results inactivation of PAR receptor, wherein said composition does not compriseeither tPA polypeptide or uPA, wherein said uPA is capable of binding tothe human uPA receptor; and (ii) a pharmaceutically acceptable carrier.5. A method for the treatment of angiogenesis-associated diseases, saidmethod comprising administering a therapeutic amount of a pharmaceuticalcomposition comprising thrombin or prothrombin to a patient diagnosedwith an angiogenesis associated disease.
 6. A method for the treatmentof angiogenesis-associated diseases, said method comprisingadministering a pharmaceutical composition comprising a compound thatmodulates PAR biological activity, wherein said treatment does notcomprise administering either tPA polypeptide or a uPA, wherein said uPAis capable of binding to the human uPA-R if said treatment alsocomprises administering captopril.
 7. A method for identifying candidatecompounds that modulate PAR biological activity, said method comprisingthe steps of: a. contacting said Protease-Activated Receptor to acandidate compound; and b. measuring binding of said compound to saidPAR receptor, wherein said binding identifies said candidate compound asa compound that is useful for modulating PAR biological activity.
 8. Amethod for the treatment of angiogenesis-associated diseases, saidmethod comprising administering a pharmaceutical composition comprisingsubstantially pure urokinase (uPA) polypeptide, wherein said polypeptideis incapable of binding to the urokinase receptor, uPA-R.
 9. A methodfor the treatment of angiogenesis-associated diseases, said methodcomprising introducing a transgene encoding a uPA polypeptide, whereinsaid uPA polypeptide is incapable of binding to uPA-R, to a cell, saidtransgene is operably linked to expression control sequences, and saidtransgene being positioned for expression in said cell.
 10. A method forthe treatment of angiogenesis-associated diseases, said methodcomprising introducing a transgene encoding a PAR polypeptide, saidtransgene is operably linked to expression control sequences, and saidtransgene being positioned for expression in said cell.
 11. A method foridentifying antiangiogenic molecules in serum plasma, said methodcomprising: a. contacting said serum plasma with a tissue protease andan ACE inhibitor; b. depleting said plasma of angiostatin; c.chromatographically separating plasma fractions; and d. determiningangiogenic potential of said fraction, wherein, inhibition ofangiogenesis identifies said fraction as antiangiogenic.
 12. The methodof claims 1, 2, 5,6, or 8-10, wherein said angiogenesis-associateddiseases is selected from the group consisting of cancer, rheumatoidarthritis, psoriasis, pyogenic granuloma, HIV Kaposi's sarcoma, diabeticretinopathy, macular degeneration, corneal graft neovascularization, andhypertrophic scarring.
 13. The method of claim 12, wherein saidangiogenesis-associated disease is cancer.
 14. The method of claims 1-4,6, 7, or 10, wherein said Protease-Activated Receptor is selected fromthe group consisting of PAR-1, PAR-3, and PAR-4.
 15. The method ofclaims 1-4, or 6, wherein said PAR-agonist or activator of the PARreceptor is selected from the group consisting of the polypeptides,SFLLRNPNDKYEPF, SFLLRN, SALLRN, GYPGKF, and SLIGKV.
 16. The method ofclaims 1-4, or 6, wherein said PAR-agonist is a monoclonal antibody. 17.The method of claim 16, wherein said monoclonal antibody modulatesPAR-receptor signaling.
 18. The method of claims 16 or 17, wherein saidmonoclonal antibody further prevents receptor internalization.
 19. Themethod of claim 5, wherein said treatment further comprisesadministering an anti-coagulant.
 20. The method of claims 1, 2, 5, 6, or8-10, wherein said treatment further comprises administering an ACEinhibitor.
 21. The method of claim 20, wherein said ACE inhibitor isselected from a group consisting of: captopril, enalapril, lisinopril,benazepril, fosinopril, ramipril, quinapril, perindopril, trandolapril,and moexipril.
 22. The method of claim 11, wherein said serum plasma ismammalian serum plasma.
 23. The method of claim 11, wherein said tissueprotease is selected from a group consisting of urokinase, tissueplasminogen activator, and streptokinase.
 24. The method of claim 11,wherein said fraction having antiangiogenic activity is further purifiedto allow for identification.
 25. The method of claim 8 or 9, whereinsaid uPA is mammalian.
 26. The method of claim 25, wherein said uPA ismouse, rat, or human.
 27. The method of claim 26, wherein said uPA ishuman uPA.
 28. The method of claim 26, wherein said human uPA furthercomprises amino acid substitutions within the Ω-loop.
 29. The method ofclaim 28, wherein said Ω-loop comprises amino acid residue substitutionson the amino acid sequences of the group consisting of the sequence²⁴tyr-²⁵phe-²⁶ser-²⁷asn-²⁸ile-²⁹his-³⁰trp in human,²⁴tyr-²⁵phe-²⁶ser-²⁷arg-²⁸ile-²⁹arg-³⁰arg in mouse, and²⁴tyr-²⁵phe-²⁶ser-²⁷ser-²⁵ile-²⁹arg-³⁰arg in rat.
 30. A pharmaceuticalcomposition comprising (i) a therapeutic amount of a uPA, wherein saiduPA is incapable of binding to the uPA-receptor; and (ii) apharmaceutically acceptable carrier.
 31. The pharmaceutical compositionof claim 30, wherein said uPA comprises amino acid substitutions withinthe Ω-loop.
 32. The pharmaceutical composition of claim 31, wherein saiduPA is mouse, rat, or human uPA..
 33. The pharmaceutical composition ofclaim 32, wherein said uPA is human uPA.
 34. The pharmaceuticalcomposition of claim 32, wherein said uPA comprises any three amino acidresidue substitutions of the sequence²⁴tyr-²⁵phe-³⁶ser-²⁷asn-²⁸ile-²⁹his-³⁰trp in human,²⁴tyr-²⁵phe-²⁶ser-²⁷arg-²⁸ile-²⁹arg-³⁰arg in mouse, and²⁴tyr-²⁵phe-²⁶ser-²⁷ser-²⁸ile²⁹arg-³⁰arg in rat.
 35. The pharmaceuticalcomposition of any of claims 30-34, wherein said pharmaceuticalcomposition is used for the treatment of an angiogenesis-associateddisease.
 36. The pharmaceutical composition of claim 35, wherein said anangiogenesis-associated disease is cancer.
 37. The pharmaceuticalcomposition of claim 36, wherein said cancer is breast cancer.
 38. Thepharmaceutical composition of any of claims 30-37, wherein saidcomposition further comprises a second therapeutic agent.
 39. Thepharmaceutical composition of claim 38, wherein said second therapeuticagent is an antiproliferative agent.
 40. The method of claim 8 or 9,wherein said method further comprises administering a therapeutic amountof an antiproliferative agent simultaneously or within 14 days of eachother in amounts sufficient to inhibit the growth of said neoplasm. 41.The method of claim 8 or 9, wherein said method further comprisesadministering a therapeutic amount of an antiproliferative agent. 42.The method of claim 9 or 10, wherein said transgene is operably linkedto tissue-specific expression control sequences.